Dehydroleucodine derivatives and uses thereof

ABSTRACT

The present invention provides dehydroleucodine derivatives. In particular, the present invention provides amine derivatives of dehydroleucodine and methods of using dehydroleucodine and the amine derivatives of dehydroleucodine to inhibit the growth of cancer cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of PCT ApplicationPCT/US2014/046389, filed Jul. 11, 2014, which claims the benefit of U.S.provisional application No. 61/845,214, filed Jul. 11, 2013, each of thedisclosures of which are hereby incorporated by reference in itsentirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under 1 DP2 OD007399-01awarded by the NIH. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to the use of dehydroleucodine and to theuse and preparation of dehydroleucodine derivatives for the treatment ofvarious types of cancers including: colon cancer, CNS cancer, melanoma,prostate cancer, acute lymphoblastic leukemia, chronic lymphocyticleukemia, acute myelogenous leukemia, chronic myeloid leukemia, andcutaneous T cell leukemia (CTCL).

BACKGROUND OF THE INVENTION

Many species of higher plants are used for healthcare purposes and alarge number of the current anticancer drugs originate from naturalsources. Given the wide variety of biologically active natural compoundsand their wide structural diversity, it may be worthwhile to continuescreening plants for compounds that could be useful as chemotherapeuticagents. Sesquiterpene lactones constitute a large and diverse group ofbiologically active plant chemicals that have been identified in severalplant families. Some sesquiterpene lactones possess anti-inflammatoryand/or antitumor activity. For example, parthenolide is highlycytotoxic, and a derivative of parthenolide is being tested in clinicaltrials as an anticancer agent. Parthenolide, however, has poorsolubility and bioavailability, thus limiting its clinical use. There isa need, therefore, for new water-soluble compounds with robust cytotoxicanticancer properties.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision ofamino derivatives of dehydroleucodine. One aspect of the disclosureencompasses a compound comprising Formula (I) or a pharmaceuticallyacceptable salt thereof:

-   -   wherein:        -   R¹ and R² are independently hydrogen, hydrocarbyl, or            substituted hydrocarbyl, or R¹ and R² together form an            optionally substituted, saturated or unsaturated,            carbocyclic or heterocyclic ring or ring system;        -   R³ is hydrogen, hydroxy, amine, cyano, halo, nitro, phospho,            sulfo, thiol, hydrocarbyl, or substituted hydrocarbyl;        -   R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are independently hydrogen,            hydroxy, amine, cyano, halo, nitro, phospho, sulfo, thiol,            hydrocarbyl, or substituted hydrocarbyl; or any pair of R⁴            and R⁵, R⁶ and R⁷, or R⁸ and R⁹ together form ═O, ═S, ═CH₂,            or ═NR^(a), wherein R^(a) is hydrogen, hydrocarbyl, or            substituted hydrocarbyl;        -   Z¹ and Z² are independently oxygen, sulfur, nitrogen, or            CH₂; and        -   is a single or double bond.

Another aspect of the present disclosure provides a method forinhibiting growth of a cancer cell. The method comprises contacting thecancer cell with an amount of a compound comprising Formula (I), or apharmaceutically acceptable salt thereof, effective to inhibit growth ofthe cancer cell. The compound comprising Formula (I):

-   -   wherein:        -   R¹ and R² are independently hydrogen, hydrocarbyl, or            substituted hydrocarbyl, or R¹ and R² together form an            optionally substituted, saturated or unsaturated,            carbocyclic or heterocyclic ring or ring system;        -   R³ is hydrogen, hydroxy, amine, cyano, halo, nitro, phospho,            sulfo, thiol, hydrocarbyl, or substituted hydrocarbyl;        -   R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are independently hydrogen,            hydroxy, amine, cyano, halo, nitro, phospho, sulfo, thiol,            hydrocarbyl, or substituted hydrocarbyl; or any pair of R⁴            and R⁵, R⁶ and R⁷, or R⁸ and R⁹ together form ═O, ═S, ═CH₂,            or ═NR^(a), wherein R^(a) is hydrogen, hydrocarbyl, or            substituted hydrocarbyl;        -   Z¹ and Z² are independently oxygen, sulfur, nitrogen, or            CH₂; and    -   is a single or double bond.

Another aspect of the present disclosure provides a method forinhibiting growth of a cancer cell. The method comprises contacting thecancer cell with an amount of dehydroleucodine, or a pharmaceuticallyacceptable salt thereof, effective to inhibit growth of the cancer cell.

Other aspects and iterations of the disclosure are described in moredetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D and FIG. 1E present the changes ingene expression in KG1 or MOLM13 cells after exposure todehydroleucodine, parthenolide, or derivatives thereof. Plotted is thefold change in expression relative to control after treatment withparthenolide-tyramine (PTL-Tyr), parthenolide (PTL), leucodine (Leuco),dehydroleucodine (DHL), dehydroleucodine-tyramine (DHL-Tyr), ordehydroleucodine-morpholine (DHL-Morp). FIG. 1A shows the expression ofHmox1 in KG1 cells. FIG. 1B plots the expression of HSPA1A in KG1 cells.FIG. 1C presents the expression of HSPH1 in KG1 cells. FIG. 1D shows theexpression of Hmox1 in MOLM13 cells. FIG. 1E presents the expression ofHSPH1 in MOLM13 cells. Error bars represent S.E.M.

FIG. 2 depicts a schematic of the synthesis of dehydroleucodine adducts.

FIG. 3 depicts the 3-D-ORTEP projection of the X-ray crystal structureof DHL-proline with 50% probability ellipsoids.

FIG. 4 depicts the 3-D-ORTEP projection of the X-ray crystal structureof DHL-morpholine with 50% probability ellipsoids.

FIG. 5A and FIG. 5B depict the conformations of DHL-morpholine. Thecrystal unit cell is composed for two distinct molecular structures ofDHL-morpholine. One conformation is (FIG. 5A) trans for C-13-N andC—H-12 bonds while the other is (FIG. 5B) gauche cis.

FIG. 6 depicts the conformational analysis of DHL-morpholine. Theresults of Grid-Search conformation analysis showed three minimums (B,D, F) and three maximums (A, C, E). The minimums corresponded with thestaggered conformations. Two of the minimums, B and F, with energies of14.60 and 14.61, respectively, correspond with the conformationsdisplayed in the crystal structure unit cell. The three maximums, A, Cand E, with energies of 21.00, 21.50 and 25.40, respectively, correspondto the eclipsed conformations.

FIG. 7A and FIG. 7B depict graphs showing that DHL is significantly lesspotent in normal peripheral blood mononuclear cells (PBMNCs) thanleukemic cells. (FIG. 7A) Viability of normal cells after treatment withDHL and its derivatives. Bone marrow and peripheral blood mononuclearcells were isolated from healthy donors and treated for 48 hours with 20μM DHL or PTL, or 50 μM Leucodine, DHL-Morpholine, DHL-Proline, orDHL-Piperidine. Cells were also treated with 10 μg/mL G. Verrucosaextract as a reference. Viability was assessed by Annexin V and 7AADstaining using flow cytometry. Each line represents a distinct sample,with viabilities calculated relative to an untreated control. (FIG. 7B)Average viability of AML cell lines compared to normal BM/PBMNCs with 20μM DHL treatment after 48 hours. The viabilities of all 8 cell linesfrom Table 5 at 20 μM DHL were averaged and graphed next to the averageviability of the 4 healthy samples from FIG. 7A. The significancebetween the two groups was calculated using the Mann-Whitney test,p=0.004.

FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D depict graphs and a western blotshowing that DHL and its derivatives induce HMOX-1 and HSPA1A, whiledownregulating NF-κB in MOLM-13 cells. (FIG. 8A) Graphicalrepresentation of the fold changes of HMOX-1 in MOLM-13 cells. Cellswere seeded at 0.5 million cells per milliliter and subject to 20 μM ofDHL, leucodine, DHL-proline, DHL-piperidine, DHL-morpholine, orparthenolide for 6 hours. RNA was then extracted and quantitative PCRwas performed. Experiments were performed in triplicates. Linesrepresent mean for each specimen, with error bars representing the SD.Fold change was calculated by the delta-delta Ct method. (FIG. 8B)Graphical representation of the fold changes of HSPA1A in MOLM-13, withthe same experimental setup and analysis as FIG. 7A. (FIG. 8C) Graphicalrepresentation of the fold changes of NF-κB in MOLM-13, with the sameexperimental setup and analysis as FIG. 7A. (FIG. 8D) Immunoblot forMOLM-13 cells after 6 hours of treatment with either DHL or PTL with 10or 20 μM. Blot was probed for phospho-p65 and p65 antibodies. Total p65is shown as the loading control.

FIG. 9A and FIG. 9B depict graphs showing that DHL and its derivativesinduce HMOX-1 and HSPA1A in MV-411 cells. (FIG. 9A) Graphicalrepresentation of the fold changes of HMOX-1 in MV-411 cells. Cells wereseeded at 0.5 million cells per milliliter and subject to 20 μM of DHL,leucodine, DHL-proline, DHL-piperidine, DHL-morpholine, or parthenolidefor 6 hours. RNA was then extracted and quantitative PCR was performed.Experiments were performed in triplicates. Lines represent mean for eachspecimen, with error bars representing the SD. Fold change wascalculated by the delta-delta Ct method. (FIG. 9B) Graphicalrepresentation of the fold changes of HSPA1A in MV-411 cells, with thesame experimental setup and analysis as FIG. 7A.

FIG. 10 depicts graphs showing that DHL and its downregulate NF-κB inMV-411 cells. Graphical representation of the fold changes of NF-κB inMV-411, with the same experimental setup and analysis as FIG. 7A.

FIG. 11A and FIG. 11B depict the structures of DHL and parthenolide,respectively. DHL is structurally more rigid than parthenolide.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are derivatives of dehydroleucodine. Primary among thevarious substituents, is the replacement of the α-methylene group of theγ-lactone ring with an amine moiety. The amine derivatives ofdehydroleucodine have improved water solubility relative todehydroleucodine. Importantly, the amine derivatives of dehydroleucodinehave anticancer activity. Also provided herein are methods of usingdehydroleucodine or derivatives of dehydroleucodine to inhibit thegrowth, proliferation, and metastasis of cancer cells. Dehydroleucodineor derivatives of dehydroleucodine, therefore, may be used to treatleukemias, colon, prostate, melanoma, central nervous system and othercancers, including multi-drug resistant cancers.

(I) Compounds Comprising Formula (I)

One aspect of the present disclosure provides a compound comprisingFormula (I) or a pharmaceutically acceptable salt thereof:

-   -   wherein:        -   R¹ and R² are independently hydrogen, hydrocarbyl, or            substituted hydrocarbyl, or R¹ and R² together form an            optionally substituted, saturated or unsaturated,            carbocyclic or heterocyclic ring or ring system;        -   R³ is hydrogen, hydroxy, amine, cyano, halo, nitro, phospho,            sulfo, thiol, hydrocarbyl, or substituted hydrocarbyl;        -   R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are independently hydrogen,            hydroxy, amine, cyano, halo, nitro, phospho, sulfo, thiol,            hydrocarbyl, or substituted hydrocarbyl; or any pair of R⁴            and R⁵, R⁶ and R⁷, or R⁸ and R⁹ together form ═O, ═S, ═CH₂,            or ═NR^(a), wherein R^(a) is hydrogen, hydrocarbyl, or            substituted hydrocarbyl;        -   Z¹ and Z² are independently oxygen, sulfur, nitrogen, or            CH₂; and        -   is a single or double bond.

In some embodiments, R¹ and R² independently may be hydrogen, alkyl,substituted alkyl, heterocyclic, substituted heterocyclic, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, or R¹ and R² together may forma heterocylic, substituted heterocyclic, heteroaryl, or substitutedheteroaryl ring or ring system. In one embodiment, R¹ and R²independently may be hydrogen, alkyl, substituted alkyl, cycloalkyl,haloalkyl, alkoxy, hydroxyalkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, arylalkyl, heterocyclic,substituted heterocyclic, heteroaryl, substituted heteroaryl,carbonylalkyl, carbonyl substituted alkyl, carbonylaryl, carbonylalkoxy,carbonylaryl, carbonylaryloxy, carbonylaminoalkyl, or carbonylaminoaryl.In another embodiment, R¹ and R² together may form —CH₂(CH₂)_(n)CH₂—where n is 0 to 5; and together with N form an optionally substitutedring, wherein the ring may be optionally fused to a cycloalkyl or arylgroup to form a bicyclic or tricyclic system, and said system may beoptionally substituted and/or optionally comprising one or moreheteroatoms. In still another embodiment, R¹ and R² together may form—CH₂(CH₂)_(n)CH₂Y—; where Y is O, S, Se, Si, P, —CO—, —SO—, —SO₂—, or—PO—; and n is 0 to 5; and together with N form an optionallysubstituted ring, wherein the ring may be optionally fused to acycloalkyl or aryl group to form a bicyclic or tricyclic ring system,and said system may be optionally substituted and/or optionallycomprising one or more heteroatoms. In a further embodiment, R¹ and R²together may form —(CH₂)_(a)—Y—(CH₂)b-; where Y is O, S, Se, Si, P,—CO—, —SO—, —SO₂—, or —PO—; and a and b are independently 1 to 4; andtogether with N form an optionally substituted ring, wherein the ringmay be optionally fused to a cycloalkyl or aryl group to form a bicyclicor tricyclic ring system, and said system may be optionally substitutedand/or optionally comprising one or more heteroatoms. In still anotherembodiment, R¹ and R² together with N may form an optionally substitutedring or ring system chosen from aziridinyl, azetidinyl, pyrrolidinyl,piperidynyl, heptamethyleneiminyl, hexamethyleneiminyl, imidazolyl,indolyl, morpholinyl, oxazinyl, piperazinyl, prolinyl, purinyl,pyrazolyl, pyrimidinyl, pyrrolyl, quinolinyl, quinuclidinyl, thiazolyl,or triazinyl.

In one embodiment, Z¹ may be oxygen and Z² may be CH₂. In still anotherembodiment, Z¹ may be nitrogen and Z² may be oxygen. In yet anotherembodiment, Z¹ may be sulfur and Z² may be oxygen. In a furtherembodiment, both Z¹ and Z² may be oxygen.

In another embodiment, the compound comprising Formula (I) may compriseFormula (Ia):

-   -   wherein:        -   R¹ and R² are as described above;        -   R³, R⁶, and R⁸ are independently hydrogen, hydroxy, amine,            cyano, halo, nitro, phospho, sulfo, thiol, hydrocarbyl, or            substituted hydrocarbyl; and        -   R⁴ and R⁵ are independently hydrogen, hydroxy, amine, cyano,            halo, nitro, phospho, sulfo, thiol, hydrocarbyl, or            substituted hydrocarbyl, or R⁴ and R⁵ together form ═O, ═S,            ═CH₂, or ═NR^(a), wherein R^(a) is hydrogen, hydrocarbyl, or            substituted hydrocarbyl.

In certain embodiments, R³, R⁵, R⁶, and R⁸ independently may behydrogen, hydroxy, alkyl, substituted alkyl, hydroxyalkyl, alkyloxy,amino, or aminoalkyl. In one embodiment, R³ and R⁸ may be C₁-C₆ alkyl,and R⁵ and R⁶ may be hydrogen. In a further embodiment, R³ and R⁸ may bemethyl, and R⁵ and R⁶ may be hydrogen. In additional embodiments, R⁴ maybe —XR^(b), wherein X is —O—, —NH—, —S—, —SO—, —SO₂—, —CO—, —CO₂— andR^(b) is hydrogen, alkyl, substituted alkyl, haloalkyl, alkoxy,hydroxyalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl,substituted heterocycloalkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, arylalkyl, heteroaryl,substituted heteroaryl, carbonylaminoalkyl, carbonylamino substitutedalkyl, carbonylaminoaryl, or carbonylamino substituted aryl.

In a further embodiment, the compound comprising Formula (I) maycomprise Formula (Ib):

-   -   wherein:        -   R¹ and R² are as described above;        -   R³, R⁴, and R⁶ are independently hydrogen, hydroxy, amine,            cyano, halo, nitro, phospho, sulfo, thiol, hydrocarbyl, or            substituted hydrocarbyl; and        -   R⁸ and R⁹ are independently are independently hydrogen,            hydroxy, amine, cyano, halo, nitro, phospho, sulfo, thiol,            hydrocarbyl, or substituted hydrocarbyl, or R⁸ and R⁹            together form ═O, ═S, ═CH₂, or ═NR^(a), wherein R^(a) is            hydrogen, hydrocarbyl, or substituted hydrocarbyl.

In various embodiments, R³, R⁴, R⁶, and R⁸ independently may behydrogen, hydroxy, alkyl, substituted alkyl, hydroxyalkyl, alkyloxy,amino, or aminoalkyl. In one embodiment, R³ may be C₁-C₆ alkyl, R⁴ andR⁶ may be hydrogen, and R⁸ may be C₁-C₆ alkyl. In a further embodiment,R³ and R⁸ may be methyl, and R⁴ and R⁶ may be hydrogen. In furtherembodiments, R⁹ may be R⁹ may be —XR^(c), wherein X is —O—, —NH—, —S—,—SO—, —SO₂—, —CO—, —CO₂— and R^(c) is hydrogen, alkyl, substitutedalkyl, hydroxyalkyl, alkenyl, substituted alkenyl, alkynyl, orsubstituted alkynyl.

In still another embodiment, the compound comprising Formula (I) maycomprise Formula (Ic):

-   -   wherein:        -   R¹ and R² are as described above;        -   R³ and R⁶ are independently hydrogen, hydroxy, amine, cyano,            halo, nitro, phospho, sulfo, thiol, hydrocarbyl, or            substituted hydrocarbyl; and        -   R⁸ and R⁹ are independently are independently hydrogen,            hydroxy, amine, cyano, halo, nitro, phospho, sulfo, thiol,            hydrocarbyl, or substituted hydrocarbyl, or R⁸ and R⁹            together form ═O, ═S, ═CH₂, or ═NR^(a), wherein R^(a) is            hydrogen, hydrocarbyl, or substituted hydrocarbyl.

In certain embodiments, R³, R⁶, and R⁸ independently may be hydrogen,hydroxy, alkyl, substituted alkyl, hydroxyalkyl, alkyloxy, amino, oraminoalkyl. In some embodiments, R³ may be C₁-C₆ alkyl, R⁶ may behydrogen or hydroxy, and R⁸ may be C₁-C₆ alkyl. In other embodiments,Wand R⁸ may be methyl, and R⁶ may be hydrogen or hydroxy. In additionalembodiments, R⁹ may be hydrogen, alkyl, substituted alkyl, cycloalkyl,haloalkyl, alkoxy, aminoalkyl, hydroxyalkyl, sulfoxyalkyl,sulfonylalkyl, thioalkyl, heterocyclic, substituted heterocyclic,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, arylalkyl, aryloxy, aminoaryl, thioaryl, sulfoxyaryl,sulfonylaryl, heteroaryl, substituted heteroaryl, cyano, carboxy,alkylcarboxylate, aryl carboxylate, carboxamide, N-alkyl carboxamide,N-aryl carboxamide, N,N-dialkyl carboxamide, N,N-diaryl carboxamide,N-alky N-aryl carboxamide, carbonyl aminoalkyl, carbonylaminosubstituted alkyl, carbonylaminoaryl, carbonylamino substituted aryl,carbonylalkoxy, or carbonylaryloxy.

In yet another embodiment, the compound comprising Formula (I) maycomprise Formula (Id):

-   -   wherein:        -   R is N-hydrocarbyl or N-substituted hydrocarbyl; and        -   R³, R⁶, and R⁸ are independently hydrogen, hydroxy, amine,            cyano, halo, nitro, phospho, sulfo, thiol, hydrocarbyl, or            substituted hydrocarbyl.

In various embodiments, R may be N-alkyl, N-cycloalkyl, N-alkenyl,N-alkynyl, N-aryl, N-substituted alkyl, N-substituted cycloalkyl,N-substituted alkenyl, N-substituted alkynyl, or N-substituted aryl. Incertain embodiments, R may be prolinyl, pipridinyl, morpholinyl,tyraminyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidynyl,heptamethyleneiminyl, hexamethyleneiminyl, imidazolyl, indolyl,morpholinyl, oxazinyl, piperazinyl, purinyl, pyrazolyl, pyrimidinyl,pyrrolyl, quinolinyl, quinuclidinyl, thiazolyl, or triazinyl. In someembodiments, R³, R⁶, and R⁸ independently may be hydrogen, hydroxy,alkyl, substituted alkyl, hydroxyalkyl, alkyloxy, amino, or aminoalkyl.In one embodiment, R³ may be C₁-C₆ alkyl, R⁶ may be hydrogen, and R⁸ maybe C₁-C₆ alkyl. In another embodiment, R³ and R⁸ may be methyl, and R⁶may be hydrogen.

The compounds comprising Formula (I) can exist in tautomeric, geometric,or stereoisomeric forms. For example, the carbons at certain positionsmay be stereogenic (or chiral). The compound comprising Formula (I) mayhave

The compounds comprising Formulas (I), (Ia), (Ib), (Ic), or (Id) canexist in tautomeric, geometric, or stereoisomeric forms. For example,the carbons at certain positions may be stereogenic (or chiral). At eachchiral center, the stereochemistry at the carbon atom is independently Ror S. The compound comprising Formula (I) can have chiral carbons at thepositions indicated below with asterisks:

The configuration of the indicated four carbons may be RRRR, RRRS, RRSR,RSRR, SRRR, RRSS, RSSR, SSRR, SRRS, SRSR, RSRS, RSSS, SRSS, SSRS, SSSR,or SSSS.

The compounds comprising Formulas (I), (Ia), (Ib), (Ic), and (Id)disclosed herein may be in the form of free bases or pharmaceuticallyacceptable salts thereof. The term “pharmaceutically acceptable salts”are salts commonly used to form alkali metal salts and to form additionsalts of free acids or free bases. The nature of the salt may vary,provided that it is pharmaceutically acceptable. Suitablepharmaceutically acceptable acid addition salts of compounds disclosedherein may be prepared from inorganic acids or organic acids.Non-limiting examples of suitable inorganic acids include hydrochloric,hydrobromic, hydroiodic, nitric, carbonic, sulfuric, perchloric, andphosphoric acid. Appropriate organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of which areformic, acetic, oxalic, propionic, succinic, glycolic, gluconic, lactic,malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, mesylic, 4-hydroxybenzoic,phenylacetic, mandelic, embonic (pamoic), methanesulfonic,ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic,toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic,hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitablepharmaceutically acceptable base addition salts of compounds disclosedherein include metallic salts made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc or organic salts made fromN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine), and procaine. All ofthese salts may be prepared by conventional means from the correspondingcompound by reacting, for example, the appropriate acid or base with theany of the compounds disclosed herein.

The compounds disclosed herein may be prepared by a variety of methods.For example, N-derived substituents may be added to the lactone moietyby contacting dehydroleucodine or a derivative thereof comprising theα-methylene-γ-lactone moiety with a secondary amine in the presence of asuitable solvent (e.g., triethylamine). The reaction mixture may beincubated at a temperature from about 10-40° C. for a period of timeranging from several hours to several days. Substituents at otherpositions may be added using well known chemical synthesis procedures.

(II) Method for Producing a Compound Comprising Formula (I)

In another embodiment, the disclosure provides a method of making thecompound comprising Formula (I). The method comprises contacting acompound comprising Formula (II) with an amine in the presence of aproton acceptor. The compound of Formula (II) comprises:

-   -   wherein, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, Z¹ and Z² may be chosen as        described in section (I).

The compound comprising Formula (II) is a dehydroleucodine or aderivative of dehydroleucodine. The process of making a compoundcomprising Formula (I) may involve the replacement of the α-methylenegroup of the γ-lactone ring with an amine moiety.

The method comprises contacting the compound comprising Formula (II)with an amine. The amine may be a primary amine (i.e., NH₂R′, wherein R′is alkyl or aryl) or a secondary amine (i.e., NHR′R″, wherein R′ and R″independently are alkyl or aryl). In some embodiments, the secondaryamine may be a cyclic amine. Non-limiting examples of suitable aminesinclude proline, piperidine, morpholine, tyramine methylamine,ethylamine, propylamine, isopropylamine, ethanolamine, aniline,dimethylamine, methylethanolamine, diphenylamine, aziridine, azetidine,pyrrolidine, heptamethyleneimine, hexamethyleneimine, imidazole, indole,oxazine, piperazine, purine, pyrazole, pyrimidine, pyrrole, quinoline,quinuclidine, thiazole, and triazine. In specific embodiments, the aminemay include proline, piperidine, morpholine, or tyramine.

The mole to mole ratio of the compound comprising Formula (II) to theamine can and will range depending upon the identity of the amine and/orthe compound of Formula (II). In general, the mole to mole ratio of thecompound comprising Formula (II) to the amine varies from about 1:1 toabout 1:40. In some embodiments, the mole to mole ratio of the compoundcomprising Formula (II) to the amine may range from about 1:10 to about1:30. In some other embodiments, the mole to mole ratio of the compoundcomprising Formula (II) to the amine may range from 1:10 to 1:30. Invarious embodiments, the mole to mole ratio of the compound comprisingFormula (II) to the amine is about 1:10, about 1:15, about 1:20, about1:25 or about 1:30. In various other embodiments, the mole to mole ratioof the compound comprising Formula (II) to the amine is 1:10, 1:15,1:20, 1:25 or 1:30. In an exemplary embodiment, the mole to mole ratioof the compound comprising Formula (II) to the amine is 1:20.

The reaction generally is carried out in the presence of a solvent.Typically, the solvent is an organic solvent. The solvent may be chosenwithout limitation from including alkane and substituted alkane solvents(including cycloalkanes) alcohol solvents, halogenated solvents,aromatic hydrocarbons, esters, ethers, ketones, and combinationsthereof. Non-limiting examples of suitable organic solvents areacetonitrile, acetone, allyl alcohol, benzene, butyl acetate,chlorobenzene, chloroform, chloromethane, cyclohexane, cyclopentane,dichloromethane (DCM), dichloroethane, diethyl ether, dimethoxyethane(DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane,ethanol, ethyl acetate, ethylene dichloride, ethylene bromide, formicacid, fluorobenzene, heptane, hexane, isobutylmethylketone, isopropanol,isopropyl acetate, N-methylpyrrolidone, methanol, methylene bromide,methylene chloride, methyl iodide, methylethylketone,methyltetrahydrofuran, pentyl acetate, propanol, n-propyl acetate,sulfolane, tetrahydrofuran (THF), tetrachloroethane, toluene,trichloroethane, water, xylene and combinations thereof. In exemplaryembodiments, one of the solvents is an alcohol solvent. In one specificembodiment, one of the solvents is ethanol.

A proton acceptor is generally added to facilitate the reaction. Theproton acceptor generally has a pKa greater than about 7, or from about7 to about 13, or more specifically from about 9 to about 11.Representative proton acceptors may include, but are not limited to,borate salts (such as, for example, NaBO₃), di- and tri-basic phosphatesalts, (such as, for example, Na₂HPO₄ and NaPO₄), bicarbonate salts,carbonate salts, hydroxides, alkoxides, (including methoxide, ethoxide,propoxide, butoxide, and pentoxide, including straight chain andbranched), and organic proton acceptors, (such as, for example,pyridine, triethylamine, N-methylmorpholine, andN,N-dimethylaminopyridine), and mixtures thereof. In some embodiments,the proton acceptor may be stabilized by a suitable counterion such aslithium, potassium, sodium, calcium, magnesium, and the like. In onespecific embodiment, the proton acceptor is triethylamine. The amount ofproton acceptor included in the reaction can and will vary, but can bereadily determined by a person of ordinary skill in the art.

The amount of time over which the reaction is conducted may also varywithin different embodiments. In some embodiments, the reaction may beconducted over a period of 2 hours to 24 hours. In particularembodiments, the reaction is carried out for about 8 hours, about 9hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours,about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18hours, about 19 hours or about 20 hours. In other embodiments, thereaction is carried out for 8 hours, 9 hours, 10 hours, 11 hours, 12hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours or 20 hours. In specific embodiments, the reaction is conductedfor about 14 hours to about 18 hours. In other specific embodiments, thereaction is conducted for 14 hours to 18 hours.

The temperature may vary over different embodiments, in some embodimentsthe temperature may range from about 20° C. to about 80° C. Inparticular embodiments the temperature may range from about 20° C. toabout 30° C., from about 30° C. to about 40° C., from about 40° C. toabout 50° C., from about 50° C. to about 60° C., from about 60° C. toabout 70° C., or from about 70° C. to about 80° C. In other particularembodiments the temperature may range from 20° C. to 30° C., from 30° C.to 40° C., from 40° C. to 50° C., from 50° C. to 60° C., from 60° C. to70° C., or from 70° C. to 80° C. In specific embodiments, thetemperature may range from about 20° C. to about 30° C. In otherspecific embodiments, the temperature may range from 20° C. to 30° C.

The synthesized compounds may be used in their crude form or they may bepurified. The compounds may be purified by any suitable method known inthe art including through column chromatography, crystallization,distillation, extraction, and the like. In one preferred embodiment, thecompounds are recrystallized from a solvent.

(III) Compositions

The present disclosure also provides pharmaceutical compositions. Thepharmaceutical composition comprises a compound comprising Formulas (I),(Ia), (Ib), (Ic), or (d), which are detailed above in section (I), as anactive ingredient and at least one pharmaceutically acceptableexcipient.

The pharmaceutically acceptable excipient may be a diluent, a binder, afiller, a buffering agent, a pH modifying agent, a disintegrant, adispersant, a preservative, a lubricant, taste-masking agent, aflavoring agent, or a coloring agent. The amount and types of excipientsutilized to form pharmaceutical compositions may be selected accordingto known principles of pharmaceutical science.

In one embodiment, the excipient may be a diluent. The diluent may becompressible (i.e., plastically deformable) or abrasively brittle.Non-limiting examples of suitable compressible diluents includemicrocrystalline cellulose (MCC), cellulose derivatives, cellulosepowder, cellulose esters (i.e., acetate and butyrate mixed esters),ethyl cellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, sodium carboxymethylcellulose, cornstarch, phosphated corn starch, pregelatinized corn starch, rice starch,potato starch, tapioca starch, starch-lactose, starch-calcium carbonate,sodium starch glycolate, glucose, fructose, lactose, lactosemonohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol,xylitol, maltodextrin, and trehalose. Non-limiting examples of suitableabrasively brittle diluents include dibasic calcium phosphate (anhydrousor dihydrate), calcium phosphate tribasic, calcium carbonate, andmagnesium carbonate.

In another embodiment, the excipient may be a binder. Suitable bindersinclude, but are not limited to, starches, pregelatinized starches,gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylamides,polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohol,polyethylene glycol, polyols, saccharides, oligosaccharides,polypeptides, oligopeptides, and combinations thereof.

In another embodiment, the excipient may be a filler. Suitable fillersinclude, but are not limited to, carbohydrates, inorganic compounds, andpolyvinylpyrrolidone. By way of non-limiting example, the filler may becalcium sulfate, both di- and tri-basic, starch, calcium carbonate,magnesium carbonate, microcrystalline cellulose, dibasic calciumphosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc,modified starches, lactose, sucrose, mannitol, or sorbitol.

In still another embodiment, the excipient may be a buffering agent.Representative examples of suitable buffering agents include, but arenot limited to, phosphates, carbonates, citrates, tris buffers, andbuffered saline salts (e.g., Tris buffered saline or phosphate bufferedsaline).

In various embodiments, the excipient may be a pH modifier. By way ofnon-limiting example, the pH modifying agent may be sodium carbonate,sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

In a further embodiment, the excipient may be a disintegrant. Thedisintegrant may be non-effervescent or effervescent. Suitable examplesof non-effervescent disintegrants include, but are not limited to,starches such as corn starch, potato starch, pregelatinized and modifiedstarches thereof, sweeteners, clays, such as bentonite,micro-crystalline cellulose, alginates, sodium starch glycolate, gumssuch as agar, guar, locust bean, karaya, pecitin, and tragacanth.Non-limiting examples of suitable effervescent disintegrants includesodium bicarbonate in combination with citric acid and sodiumbicarbonate in combination with tartaric acid.

In yet another embodiment, the excipient may be a dispersant ordispersing enhancing agent. Suitable dispersants may include, but arenot limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum,kaolin, bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose.

In another alternate embodiment, the excipient may be a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate,citric acid, sodium citrate; chelators such as EDTA or EGTA; andantimicrobials, such as parabens, chlorobutanol, or phenol.

In a further embodiment, the excipient may be a lubricant. Non-limitingexamples of suitable lubricants include minerals such as talc or silica;and fats such as vegetable stearin, magnesium stearate or stearic acid.

In yet another embodiment, the excipient may be a taste-masking agent.Taste-masking materials include cellulose ethers; polyethylene glycols;polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers;monoglycerides or triglycerides; acrylic polymers; mixtures of acrylicpolymers with cellulose ethers; cellulose acetate phthalate; andcombinations thereof.

In an alternate embodiment, the excipient may be a flavoring agent.Flavoring agents may be chosen from synthetic flavor oils and flavoringaromatics and/or natural oils, extracts from plants, leaves, flowers,fruits, and combinations thereof.

In still a further embodiment, the excipient may be a coloring agent.Suitable color additives include, but are not limited to, food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drugand cosmetic colors (Ext. D&C).

The weight fraction of the excipient or combination of excipients in thecomposition may be about 99% or less, about 97% or less, about 95% orless, about 90% or less, about 85% or less, about 80% or less, about 75%or less, about 70% or less, about 65% or less, about 60% or less, about55% or less, about 50% or less, about 45% or less, about 40% or less,about 35% or less, about 30% or less, about 25% or less, about 20% orless, about 15% or less, about 10% or less, about 5% or less, about 2%,or about 1% or less of the total weight of the composition.

The composition can be formulated into various dosage forms andadministered by a number of different means that will deliver atherapeutically effective amount of the active ingredient. Suchcompositions can be administered orally, parenterally, or topically indosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants, and vehicles asdesired. Topical administration may also involve the use of transdermaladministration such as transdermal patches or iontophoresis devices. Theterm parenteral as used herein includes subcutaneous, intravenous,intramuscular, or intrasternal injection, or infusion techniques.Formulation of drugs is discussed in, for example, Gennaro, A. R.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.(18^(th) ed, 1995), and Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980).

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, powders, pellets, and granules. In such solid dosageforms, the active ingredient is ordinarily combined with one or morepharmaceutically acceptable excipients, examples of which are detailedabove. Oral preparations may also be administered as aqueoussuspensions, elixirs, or syrups. For these, the active ingredient may becombined with various sweetening or flavoring agents, coloring agents,and, if so desired, emulsifying and/or suspending agents, as well asdiluents such as water, ethanol, glycerin, and combinations thereof.

For parenteral administration (including subcutaneous, intradermal,intravenous, intramuscular, and intraperitoneal), the preparation may bean aqueous or an oil-based solution. Aqueous solutions may include asterile diluent such as water, saline solution, a pharmaceuticallyacceptable polyol such as glycerol, propylene glycol, or other syntheticsolvents; an antibacterial and/or antifungal agent such as benzylalcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and thelike; an antioxidant such as ascorbic acid or sodium bisulfite; achelating agent such as etheylenediaminetetraacetic acid; a buffer suchas acetate, citrate, or phosphate; and/or an agent for the adjustment oftonicity such as sodium chloride, dextrose, or a polyalcohol such asmannitol or sorbitol. The pH of the aqueous solution may be adjustedwith acids or bases such as hydrochloric acid or sodium hydroxide.Oil-based solutions or suspensions may further comprise sesame, peanut,olive oil, or mineral oil.

For topical (e.g., transdermal or transmucosal) administration,penetrants appropriate to the barrier to be permeated are generallyincluded in the preparation. Transmucosal administration may beaccomplished through the use of nasal sprays, aerosol sprays, tablets,or suppositories, and transdermal administration may be via ointments,salves, gels, patches, or creams as generally known in the art.

In certain embodiments, a composition comprising DHL or a compound ofthe invention is encapsulated in a suitable vehicle to either aid in thedelivery of the compound to target cells, to increase the stability ofthe composition, or to minimize potential toxicity of the composition.As will be appreciated by a skilled artisan, a variety of vehicles aresuitable for delivering a composition of the present invention.Non-limiting examples of suitable structured fluid delivery systems mayinclude nanoparticles, liposomes, microemulsions, micelles, dendrimersand other phospholipid-containing systems. Methods of incorporatingcompositions into delivery vehicles are known in the art.

In one alternative embodiment, a liposome delivery vehicle may beutilized. Liposomes, depending upon the embodiment, are suitable fordelivery of the compound of the invention in view of their structuraland chemical properties. Generally speaking, liposomes are sphericalvesicles with a phospholipid bilayer membrane. The lipid bilayer of aliposome may fuse with other bilayers (e.g., the cell membrane), thusdelivering the contents of the liposome to cells. In this manner, thecompound of the invention may be selectively delivered to a cell byencapsulation in a liposome that fuses with the targeted cell'smembrane.

Liposomes may be comprised of a variety of different types ofphospholipids having varying hydrocarbon chain lengths. Phospholipidsgenerally comprise two fatty acids linked through glycerol phosphate toone of a variety of polar groups. Suitable phospholipids includephosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol(PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG),phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fattyacid chains comprising the phospholipids may range from about 6 to about26 carbon atoms in length, and the lipid chains may be saturated orunsaturated. Suitable fatty acid chains include (common name presentedin parentheses) n-dodecanoate (laurate), n-tretradecanoate (myristate),n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate(arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate),cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate),cis,cis-9,12-octadecandienoate (linoleate), all cis-9, 12,15-octadecatrienoate (linolenate), and allcis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acidchains of a phospholipid may be identical or different. Acceptablephospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS,distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl,oleoyl PS, palmitoyl, linolenyl PS, and the like.

The phospholipids may come from any natural source, and, as such, maycomprise a mixture of phospholipids. For example, egg yolk is rich inPC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brainor spinal cord is enriched in PS. Phospholipids may come from syntheticsources too. Mixtures of phospholipids having a varied ratio ofindividual phospholipids may be used. Mixtures of differentphospholipids may result in liposome compositions having advantageousactivity or stability of activity properties. The above mentionedphospholipids may be mixed, in optimal ratios with cationic lipids, suchas N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride,1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,3,3′-deheptyloxacarbocyanine iodide,1,1′-dedodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,1,1′-dioleyl-3,3,3′,3′-tetramethylindo carbocyanine methanesulfonate,N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or1,1,-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate.

Liposomes may optionally comprise sphingolipids, in which spingosine isthe structural counterpart of glycerol and one of the one fatty acids ofa phosphoglyceride, or cholesterol, a major component of animal cellmembranes. Liposomes may optionally, contain pegylated lipids, which arelipids covalently linked to polymers of polyethylene glycol (PEG). PEGsmay range in size from about 500 to about 10,000 daltons.

Liposomes may further comprise a suitable solvent. The solvent may be anorganic solvent or an inorganic solvent. Suitable solvents include, butare not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone,N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide,tetrahydrofuran, or combinations thereof.

Liposomes carrying the compound of the invention (i.e., having at leastone methionine compound) may be prepared by any known method ofpreparing liposomes for drug delivery, such as, for example, detailed inU.S. Pat. Nos. 4,241,046, 4,394,448, 4,529,561, 4,755,388, 4,828,837,4,925,661, 4,954,345, 4,957,735, 5,043,164, 5,064,655, 5,077,211 and5,264,618, the disclosures of which are hereby incorporated by referencein their entirety. For example, liposomes may be prepared by sonicatinglipids in an aqueous solution, solvent injection, lipid hydration,reverse evaporation, or freeze drying by repeated freezing and thawing.In a preferred embodiment the liposomes are formed by sonication. Theliposomes may be multilamellar, which have many layers like an onion, orunilamellar. The liposomes may be large or small. Continued high-shearsonication tends to form smaller unilamellar liposomes.

As would be apparent to one of ordinary skill, all of the parametersthat govern liposome formation may be varied. These parameters include,but are not limited to, temperature, pH, concentration of methioninecompound, concentration and composition of lipid, concentration ofmultivalent cations, rate of mixing, presence of and concentration ofsolvent.

In another embodiment, a composition of the invention may be deliveredto a cell as a microemulsion. Microemulsions are generally clear,thermodynamically stable solutions comprising an aqueous solution, asurfactant, and “oil.” The “oil” in this case, is the supercriticalfluid phase. The surfactant rests at the oil-water interface. Any of avariety of surfactants are suitable for use in microemulsionformulations including those described herein or otherwise known in theart. The aqueous microdomains suitable for use in the inventiongenerally will have characteristic structural dimensions from about 5 nmto about 100 nm. Aggregates of this size are poor scatterers of visiblelight and hence, these solutions are optically clear. As will beappreciated by a skilled artisan, microemulsions can and will have amultitude of different microscopic structures including sphere, rod, ordisc shaped aggregates. In one embodiment, the structure may bemicelles, which are the simplest microemulsion structures that aregenerally spherical or cylindrical objects. Micelles are like drops ofoil in water, and reverse micelles are like drops of water in oil. In analternative embodiment, the microemulsion structure is the lamellae. Itcomprises consecutive layers of water and oil separated by layers ofsurfactant. The “oil” of microemulsions optimally comprisesphospholipids. Any of the phospholipids detailed above for liposomes aresuitable for embodiments directed to microemulsions. The composition ofthe invention may be encapsulated in a microemulsion by any methodgenerally known in the art.

In yet another embodiment, a composition of the invention may bedelivered in a dendritic macromolecule, or a dendrimer. Generallyspeaking, a dendrimer is a branched tree-like molecule, in which eachbranch is an interlinked chain of molecules that divides into two newbranches (molecules) after a certain length. This branching continuesuntil the branches (molecules) become so densely packed that the canopyforms a globe. Generally, the properties of dendrimers are determined bythe functional goups at their surface. For example, hydrophilic endgroups, such as carboxyl groups, would typically make a water-solubledendrimer. Alternatively, phospholipids may be incorporated in thesurface of a dendrimer to facilitate absorption across the skin. Any ofthe phospholipids detailed for use in liposome embodiments are suitablefor use in dendrimer embodiments. Any method generally known in the artmay be utilized to make dendrimers and to encapsulate compositions ofthe invention therein. For example, dendrimers may be produced by aniterative sequence of reaction steps, in which each additional iterationleads to a higher order dendrimer. Consequently, they have a regular,highly branched 3D structure, with nearly uniform size and shape.Furthermore, the final size of a dendrimer is typically controlled bythe number of iterative steps used during synthesis. A variety ofdendrimer sizes are suitable for use in the invention. Generally, thesize of dendrimers may range from about 1 nm to about 100 nm.

(IV) Methods for Inhibiting Cancer Cell Growth

A further aspect of the present disclosure provides a method forinhibiting growth of a cancer cell. Cancer cell growth includes cellproliferation and cell metastasis. The method comprises contacting thecancer cell with an effective amount of a compound comprising Formulas(I), (Ia), (Ib), (Ic), or (Id), or a pharmaceutically acceptable saltthereof, wherein the amount is effective to inhibit growth of the cancercell. Compounds comprising Formulas (I), (Ia), (Ib), (Ic), and (Id) aredetailed above in section (I). In some embodiments, the compoundcomprising Formulas (I), (Ia), (Ib), (Ic), or (Id) is used as part of acomposition, examples of which are detailed above in section (III).

In another embodiment, the method comprises contacting the cancer cellwith an effective amount of dehydroleucodine or a pharmaceuticallyacceptable salt thereof, wherein the amount is effective to inhibitgrowth of the cancer cell. In some embodiments, dehydroleucodine is usedas part of a composition, examples of which are detailed above insection (III).

(a) Contacting the Cell

In some embodiments, the cancer cell may be in vitro. The cancer cellmay be an established, commercially-available cancer cell line (e.g.,American Type Culture Collection (ATCC), Manassas, Va.). The cancer cellline may be derived from a blood cancer or a solid tumor. The cancercell line may be a human cell line or a mammalian cell line. In aspecific embodiment, the cancer cell line may be derived from a bloodcancer. In one exemplary embodiment, the cancer cell line may be derivedfrom a leukemic cell. The leukemic cell may be an acute myeloid leukemiacell, a chronic myeloid leukemia cell, an acute lymphocytic leukemiacell, a chronic lymphocytic leukemia cell, a cutaneous T cell leukemia,or another type of leukemia cell. In some embodiments, the cancer cellline may be a leukemia cell line such as Kasumi-1, KCL22, KG-1, MV4-11,MOLM-13, TF-1, THP-1, TUR, HL-60, U937, CCRF-CEM, K-562 or RPMI-8226. Inother embodiments, the cancer cell line may be a hematopoietic orlymphoid cell line. Non-limiting examples of hematopoietic or lymphoidcell lines include 380, 697, A3-KAW, A3/KAW, A4-Fuk, A4/Fuk, ALL-PO,ALL-SIL, AML-193, AMO-1, ARH-77, ATN-1, BALL-1, BC-3, BCP-1, BDCM,BE-13, BL-41, BL-70, BV-173, C8166, CA46, CCRF-CEM, CI-1, CMK, CMK-11-5,CMK-86, CML-T1, COLO 775, COLO-677, CTB-1, CTV-1, Daudi, DB, DEL, DG-75,DND-41, DOHH-2, EB1, EB2, EHEB, EJM, EM-2, EOL-1, EoL-1-cell, F-36P,GA-10, GA-10-Clone-4, GDM-1, GR-ST, GRANTA-519, H9, HAL-01, HD-MY-Z,HDLM-2, HEL, HEL 92.1.7, HH, HL-60, HPB-ALL, Hs 604.T, Hs 611.T, Hs616.T, Hs 751.T, HT, HTK-, HuNS1, HuT 102, HuT 78, IM-9, J-RT3-T3-5,JeKo-1, JiyoyeP-2003, JJN-3, JK-1, JM1, JURKAT, JURL-MK1, JVM-2, JVM-3,K-562, K052, KARPAS-299, KARPAS-422, KARPAS-45, KARPAS-620, KASUMI-1,KASUMI-2, Kasumi-6, KCL-22, KE-37, KE-97, KG-1, KHM-1B, Ki-JK, KM-H2,KMM-1, KMOE-2, KMS-11, KMS-12-BM, KMS-12-PE, KMS-18, KMS-20, KMS-21BM,KMS-26, KMS-27, KMS-28BM, KMS-34, K052, KOPN-8, KU812, KY821, KYO-1,L-1236, L-363, L-428, L-540, LAMA-84, LC4-1, Loucy, LOUCY, LP-1, M-07e,MC-CAR, MC116, ME-1, MEC-1, MEC-2, MEG-01, MHH-CALL-2, MHH-CALL-3,MHH-CALL-4, MHH-PREB-1, Mino, MJ, ML-2, MLMA, MM1-S, MN-60, MOLM-13,MOLM-16, MOLM-6, MOLP-2, MOLP-8, MOLT-13, MOLT-16, MOLT-4, MONO-MAC-1,MONO-MAC-6, MOTN-1, MUTZ-1, MUTZ-3, MUTZ-5, MV-4-11, NALM-1, NALM-19,NALM-6, NAMALWA, NB-4, NCI-H929, NCO2, NKM-1, NOMO-1, NU-DHL-1,NU-DUL-1, OCI-AML2, OCI-AML3, OCI-AML5, OCI-LY-19, OCI-LY10, OCI-LY3,OCI-M1, OPM-2, P12-ICHIKAWA, P30-OHK, P31-FUJ, P31/FUJ, P3HR-1, PCM6,PEER, PF-382, Pfeiffer, PL-21, Raji, Ramos-2G6-4C10, RCH-ACV, REC-1,Reh, REH, RI-1, RL, RPMI 8226, RPMI-8226, RPMI-8402, RS4-11, “RS4;11”,SEM, Set-2, SIG-M5, SK-MM-2, SKM-1, SR, SR-786, ST486, SU-DHL-1,SU-DHL-10, SU-DHL-4, SU-DHL-5, SU-DHL-6, SU-DHL-8, SUP-B15, SUP-B8,SUP-HD1, SUP-M2, SUP-T1, SUP-T11, TALL-1, TF-1, THP-1, TO 175.T, Toledo,TUR, U-266, U-698-M, U-937, U266B1, UT-7, WSU-DLCL2, and WSU-NHL.

In another exemplary embodiment, the cancer cell line may be derivedfrom a solid tumor cell. The solid tumor cell may be a colon cancercell, a prostate cancer cell, a central nervous system cancer cell, amelanoma cell, or another type of solid tumor cell. In a specificembodiment, the solid tumor cell may be a colon cancer cell, a prostatecancer cell, or a central nervous system (CNS) cancer cell. In someembodiments, the cancer cell line may be a human solid tumor cell linesuch as HCT-116, SW-620, SNB-75, U251, PC-3, or DU-145. In otherembodiments, the cancer cell line may be any colon cancer cell line.Non-limiting examples of colon cancer cell lines include C2BBe1, CaR-1,CCK-81, CL-11, CL-14, CL-34, CL-40, CoCM-1, COLO 201, COLO 205,COLO-205, COLO-320, COLO-320-HSR, COLO-678, COLO-741, CW-2, DLD-1, GP2d,GP5d, HCC-56, HCC2998, HCT 116, HCT-116, HCT-15, HCT-8, HRT-18, Hs255.T, Hs 675.T, Hs 698.T, HT-29, HT115, HT55, KM12, LoVo, LS 180,LS-1034, LS-123, LS-174T, LS-411N, LS-513, LS1034, LS123, LS411N, LS513,MDST8, NCI-HSO8, NCI-H630, NCI-H716, NCI-H747, OUMS-23, RCM-1, RKO,SK-CO-1, SNU-1033, SNU-1040, SNU-1197, SNU-175, SNU-283, SNU-407,SNU-503, SNU-61, SNU-81, SNU-C1, SNU-C2A, SNU-C2B, SNU-C4, SNU-05,SW1116, SW1417, SW1463, SW403, SW48, SW480, SW620, SW837, SW948, andT84. In still other embodiments, the cancer cell line may be anyprostate cancer cell line. Non-limiting examples of prostate cancer celllines include 22Rv1, 22RV1, DU 145, DU-145, LNCaP, clone FGC,LNCaP-Clone-FGC, MDA PCa 2b, NCI-H660, PC-3, PrEC LH, and VCaP. In stillother embodiments, the cancer cell line may be any central nervoussystem cancer cell line. Non-limiting examples of central nervous systemcancer cell lines include 1321N1, 42-MG-BA, 8-MG-BA, A1207, A172, AM-38,Becker, CAS-1, CCF-STTG1, CH-157MN, D-245MG, D-247MG, D-263MG, D-336MG,D-392MG, D-397MG, D-423MG, D-502MG, D-538MG, D-542MG, D-566MG, D283 Med,D341 Med, Daoy, DBTRG-05MG, DK-MG, F5, GAMG, GB-1, GI-1, GMS-10, GOS-3,H4, Hs 683, IOMM-Lee, KALS-1, KG-1-C, KINGS-1, KNS-42, KNS-60, KNS-81,KNS-81-FD, KS-1, LN-18, LN-215, LN-229, LN-235, LN-319, LN-340, LN-405,LN-428, LN-443, LN-464, LN382, LNZ308, M059J, M059K, MOG-G-CCM,MOG-G-UVW, NMC-G1, no-10, no-11, ONS-76, PFSK-1, SF-172, SF-295, SF126,SF268, SF295, SF539, SF767, SK-MG-1, SNB-19, SNB19, SNB75, SNU-1105,SNU-201, SNU-466, SNU-489, SNU-626, SNU-738, SW 1088, SW 1783, SW 1088,SW 1783, T98G, TM-31, U-118 MG, U-118-MG, U-138 MG, U-178, U-251 MG,U-87 MG, U-87-MG, U251, U343, YH-13, YKG-1, and YKG1.

In other embodiments, the cancer cell may be in vivo; i.e., the cell maybe disposed in a subject. In such embodiments, the cancer cell iscontacted with the compound comprising Formula (I) by administering thecompound comprising Formula (I) to the subject. In another embodiment,the cancer cell is contacted with dehydroleucodine by administeringdehydroleucodine to the subject. In some embodiments, the subject may bea human. In other embodiments, the subject may be a non-human animal.Non-limiting examples of non-human animals include companion animals(e.g., cats, dogs, horses, rabbits, gerbils), agricultural animals(e.g., cows, pigs, sheep, goats, fowl), research animals (e.g., rats,mice, rabbits, primates), and zoo animals (e.g., lions, tiger,elephants, and the like).

The cancer cell disposed in the subject may be a blood cancer cell(e.g., leukemia, lymphoma, myeloma) or a solid tumor cancer cell. Thecancer may be primary or metastatic; early stage or late stage; and/orthe tumor may be malignant or benign. Non-limiting cancers includebladder cancer, bone cancer, brain cancer, breast cancer, centralnervous system cancer, cervical cancer, colon cancer, colorectal cancer,duodenal cancer, endometrial cancer, esophageal cancer, eye cancer,gallbladder cancer, germ cell cancer, kidney cancer, larynx cancer,leukemia, liver cancer, lymphoma, lung cancer, melanoma, mouth/throatcancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer,testicular cancer, thyroid cancer, vaginal cancer, and drug resistantcancers. In one exemplary embodiment, the cancer cell may be a leukemia.The leukemia may be an acute lymphocytic (lymphoblastic) leukemia, achronic lymphocytic leukemia, an acute myeloid leukemia, a chronicmyeloid leukemia, a hairy cell leukemia, a T-cell prolymphocyticleukemia, a large granular lymphocytic leukemia, or an adult T-cellleukemia. In another exemplary embodiment, the cancer cell may be acolon cancer. In still another exemplary embodiment, the cancer cell maybe a prostate cancer. In still yet another exemplary embodiment, thecancer cell may be a CNS cancer. DHL may be especially useful in CNScancer because of its experimentally determined Log P octanol/water of2.33 (±0.16) that is close to the optimum to penetrate the blood-brainbarrier (Log P=2)

Dehydroleucodine or the compound comprising Formulas (I), (Ia), (Ib),(Ic), or (Id) may be administered to the subject orally (as a solid or aliquid), parenterally (which includes intramuscular, intravenous,intradermal, intraperitoneal, and subcutaneous), or topically (whichincludes transmucosal and transdermal). An effective amount of thecompound can be determined by a skilled practitioner in view of desireddosages and potential side effects of the compound.

Dehydroleucodine or the compound comprising Formulas (I), (Ia), (Ib),(Ic), or (Id) may be administered once or administered repeatedly to thesubject. Repeated administrations may be at regular intervals of 2hours, 6 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 30 days, andso forth.

(b) Inhibiting Cancer Cell Growth

Following contact with an effective amount of the compound comprisingFormulas (I), (Ia), (Ib), (Ic), or (Id), growth of the cancer cell isinhibited. Additionally, following contact with an effective amount ofdehydroleucodine, growth of the cancer cell is inhibited. Cell growth orproliferation can be measured in cells grown in vitro using standardcell viability or cell cytotoxicity assays (e.g., based on DNA content,cell permeability, etc.) in combination with cell counting methods(e.g., flow cytometry, optical density). Cell growth or proliferationcan be measured in vivo using imaging procedures and/or moleculardiagnostic indicators.

In an embodiment, contact with an effective amount of the compoundcomprising Formulas (I), (Ia), (Ib), (Ic), or (Id) selectively inhibitsgrowth of cancer cells. As such, a compound comprising Formulas (I),(Ia), (Ib), (Ic), or (Id) does not appreciably kill non-cancer cells atthe same concentration. Accordingly, more than 50% of non-cancer cellsremain viable following contact with a compound comprising Formulas (I),(Ia), (Ib), (Ic), or (Id) at the same concentration. For example about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95% or about 100% of non-cancer cells remainviable following contact with a compound comprising Formulas (I), (Ia),(Ib), (Ic), or (Id) at the same concentration. Or, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 100% of non-cancer cells remain viablefollowing contact with a compound comprising Formulas (I), (Ia), (Ib),(Ic), or (Id) at the same concentration.

In another embodiment, contact with an effective amount ofdehydroleucodine selectively inhibits growth of cancer cells. As such,dehydroleucodine does not appreciably kill non-cancer cells at the sameconcentration. Accordingly, more than 50% of non-cancer cells remainviable following contact with dehydroleucodine at the sameconcentration. For example about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95% orabout 100% of non-cancer cells remain viable following contact withdehydroleucodine at the same concentration. Or, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 100% of non-cancer cells remain viablefollowing contact with dehydroleucodine at the same concentration.

In various embodiments, cancer cell growth may be inhibited about0.5-fold, about 1-fold, about 2-fold, about 3-fold, about 4-fold, about5-fold, about 8-fold, about 10-fold, or more than 10-fold relative to areference value. In various other embodiments, cancer cell growth may beinhibited 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold,10-fold, or more than 10-fold relative to a reference value. In otherembodiments, cancer cell growth may be inhibited to such a degree thatthe cell undergoes cell death (via apoptosis or necrosis). Any suitablereference value known in the art may be used. For example, a suitablereference value may be cancer cell growth in a sample that has not beencontacted with dehydroleucodine or a compound comprising Formulas (I),(Ia), (Ib), (Ic), or (Id). In another example, a suitable referencevalue may be the baseline growth rate of the cells as determined bymethods known in the art. In another example, a suitable reference valuemay be a measurement of the number of cancer cells in a reference sampleobtained from the same subject. For example, when monitoring theeffectiveness of a therapy or efficacy of dehydroleucodine or a compoundcomprising Formulas (I), (Ia), (Ib), (Ic), or (Id), a reference samplemay be a sample obtained from a subject before therapy or administrationof dehydroleucodine or the compound comprising Formulas (I), (Ia), (Ib),(Ic), or (Id) began.

In an embodiment, contact with an effective amount of the compoundcomprising Formulas (I), (Ia), (Ib), (Ic), or (Id) inhibits activationof NF-κB. In another embodiment, contact with an effective amount ofdehydroleucodine inhibits activation of NF-κB. Inhibition of activationof NF-κB may induce apoptosis. As such, the inhibition of activation ofNF-κB may be measured in vitro using standard cell viability or cellcytotoxicity assays in combination with cell counting methods asdescribed above. Or, the inhibition of activation of NF-κB may bemeasured in vivo using imaging procedures and/or molecular diagnosticindicators. Inhibition of activation of NF-κB may also be measured bymeasuring nucleic acid expression of NF-κB. Methods to measure nucleicacid expression are known in the art and may include PCR, quantitativePCR, RT-PCR, qRT-PCR, microarray or array.

In various embodiments, expression of NF-κB may be reduced about0.5-fold, about 1-fold, about 2-fold, about 3-fold, about 4-fold, about5-fold, about 8-fold, about 10-fold, or more than 10-fold relative to areference value. In various other embodiments, cancer cell growth may beinhibited 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold,10-fold, or more than 10-fold relative to a reference value. Anysuitable reference value known in the art may be used. For example, asuitable reference value may be the expression of NF-κB in a sample thathas not been contacted with dehydroleucodine or a compound comprisingFormulas (I), (Ia), (Ib), (Ic), or (Id). In another example, a suitablereference value may be the expression of NF-κB in a subject, or group ofsubjects, of the same species that has no clinically detectable symptomof cancer. In another example, a suitable reference value may beexpression of NF-κB in a subject, or group of subjects, of the samespecies that has no clinically detectable cancer. In another example, asuitable reference value may be the background signal of the assay asdetermined by methods known in the art. In another example, a suitablereference value may be a measurement of the expression of NF-κB in areference sample obtained from the same subject. The reference samplemay be obtained from a subject when the subject had no clinicallydetectable symptom of cancer. A skilled artisan will appreciate that itis not always possible or desirable to obtain a reference sample from asubject when the subject is otherwise healthy. For example, whenmonitoring the effectiveness of a therapy or efficacy ofdehydroleucodine or a compound comprising Formulas (I), (Ia), (Ib),(Ic), or (Id), a reference sample may be a sample obtained from asubject before therapy or administration of dehydroleucodine or thecompound comprising Formulas (I), (Ia), (Ib), (Ic), or (Id) began. In anadditional example, a suitable reference sample may be from anindividual or group of individuals that has been shown not to havecancer.

In another embodiment, contact with an effective amount of the compoundcomprising Formulas (I), (Ia), (Ib), (Ic), or (Id) induces oxidativestress. Alternatively, contact with an effective amount ofdehydroleucodine induces oxidative stress. Induction of oxidative stressmay result in cell death, apoptosis and/or necrosis. As such, theinduction of oxidative stress may be measured in vitro using standardcell viability or cell cytotoxicity assays in combination with cellcounting methods as described above. Or, the induction of oxidativestress may be measured in vivo using imaging procedures and/or moleculardiagnostic indicators. Induction of oxidative stress may also bemeasured by measuring nucleic acid expression of markers of oxidativestress. A skilled artisan would be able to markers of oxidative stress.In an exemplary embodiment markers of oxidative stress may be, but notlimited to, HMOX1, HSPA1A and HSPH1. As such, induction of oxidativestress may be measured by measuring nucleic acid expression of HMOX1,HSPA1A and/or HSPH1. Methods to measure nucleic acid expression areknown in the art and may include PCR, quantitative PCR, RT-PCR, qRT-PCR,microarray or array.

In various embodiments, expression of HMOX1, HSPA1A and/or HSPH1 may beinduced about 0.5-fold, about 1-fold, about 2-fold, about 3-fold, about4-fold, about 5-fold, about 8-fold, about 10-fold, or more than 10-foldrelative to a reference value. In various other embodiments, expressionof HMOX1, HSPA1A and/or HSPH1 may be induced 0.5-fold, 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 8-fold, 10-fold, or more than 10-fold relativeto a reference value. In other embodiments, expression of HMOX1, HSPA1Aand/or HSPH1 may be induced about 10-fold, about 20-fold, about 30-fold,about 40-fold, about 50-fold, about 60-fold, about 70-fold, about80-fold, about 90-fold, about 100-fold or more than 100-fold relative toa reference value. In still other embodiments, expression of HMOX1,HSPA1A and/or HSPH1 may be induced 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more than100-fold relative to a reference value. In still yet other embodiments,expression of HMOX1, HSPA1A and/or HSPH1 may be induced about 100-fold,about 1000-fold, about 2000-fold, about 3000-fold, about 4000-fold,about 5000-fold, or more than 5000-fold relative to a reference value.In different embodiments, expression of HMOX1, HSPA1A and/or HSPH1 maybe induced 100-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold,5000-fold, or more than 5000-fold relative to a reference value. Anysuitable reference value known in the art may be used. For example, asuitable reference value may be the expression of HMOX1, HSPA1A and/orHSPH1 in a sample that has not been contacted with a compound comprisingFormulas (I), (Ia), (Ib), (Ic), or (Id). In another example, a suitablereference value may be the expression of HMOX1, HSPA1A and/or HSPH1 in asubject, or group of subjects, of the same species that has noclinically detectable symptom of cancer. In another example, a suitablereference value may be expression of HMOX1, HSPA1A and/or HSPH1 in asubject, or group of subjects, of the same species that has noclinically detectable cancer. In another example, a suitable referencevalue may be the background signal of the assay as determined by methodsknown in the art. In another example, a suitable reference value may bea measurement of the expression of HMOX1, HSPA1A and/or HSPH1 in areference sample obtained from the same subject. The reference samplemay be obtained from a subject when the subject had no clinicallydetectable symptom of cancer. A skilled artisan will appreciate that itis not always possible or desirable to obtain a reference sample from asubject when the subject is otherwise healthy. For example, whenmonitoring the effectiveness of a therapy or efficacy ofdehydroleucodine or a compound comprising Formulas (I), (Ia), (Ib),(Ic), or (Id), a reference sample may be a sample obtained from asubject before therapy or administration of dehydroleucodine or thecompound comprising Formulas (I), (Ia), (Ib), (Ic), or (Id) began. In anadditional example, a suitable reference sample may be from anindividual or group of individuals that has been shown not to havecancer.

(c) Optional Contact

In certain embodiments, the method may further comprise contacting thecell with at least one chemotherapeutic agent and/or a radiotherapeuticagent. The chemotherapeutic agent and/or radiotherapeutic agent may beadministered concurrently or sequentially with dehydroleucodine or thecompound comprising Formulas (I), (Ia), (Ib), (Ic), or (Id).

The chemotherapeutic agent may be an alkylating agent, ananti-metabolite, an anti-tumor antibiotic, an anti-cytoskeletal agent, atopoisomerase inhibitor, an anti-hormonal agent, a targeted therapeuticagent, or a combination thereof. Non-limiting examples of suitablealkylating agents include altretamine, benzodopa, busulfan, carboplatin,carboquone, carmustine (BCNU), chlorambucil, chlornaphazine,cholophosphamide, chlorozotocin, cisplatin, cyclosphosphamide,dacarbazine (DTIC), estramustine, fotemustine, ifosfamide, improsulfan,lomustine (CCNU), mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, meturedopa, nimustine, novembichin, phenesterine, piposulfan,prednimustine, ranimustine; temozolomide, thiotepa, triethylenemelamine,trietylenephosphoramide, triethylenethiophosphaoramide,trimethylolomelamine, trofosfamide, uracil mustard and uredopa. Suitableanti-metabolites include, but are not limited to aminopterin,ancitabine, azacitidine, 6-azauridine, capecitabine, carmofur(1-hexylcarbomoyl-5-fluorouracil), cladribine, cytarabine or cytosinearabinoside (Ara-C), dideoxyuridine, denopterin, doxifluridine,enocitabine, floxuridine, fludarabine, 5-fluorouracil, gemcetabine,hydroxyurea, leucovorin (folinic acid), 6-mercaptopurine, methotrexate,pemetrexed, pteropterin, thiamiprine, trimetrexate, and thioguanine.Non-limiting examples of suitable anti-tumor antibiotics includeaclacinomysin, actinomycins, adriamycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mithramycin, mycophenolic acid,nogalamycin, olivomycins, peplomycin, plicamycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, valrubicin, ubenimex, zinostatin, and zorubicin.Non-limiting examples of suitable anti-cytoskeletal agents includecolchicines, docetaxel, macromycin, paclitaxel, vinblastine,vincristine, vindesine, and vinorelbine. Suitable topoisomeraseinhibitors include, but are not limited to, amsacrine, etoposide(VP-16), irinotecan, mitoxantrone, RFS 2000, teniposide, and topotecan.Non-limiting examples of suitable anti-hormonal agents such asaminoglutethimide, aromatase inhibiting 4(5)-imidazoles, bicalutamide,finasteride, flutamide, goserelin, 4-hydroxytamoxifen, keoxifene,leuprolide, LY117018, mitotane, nilutamide, onapristone, raloxifene,tamoxifen, toremifene, and trilostane. Examples of targeted therapeuticagents include, without limit, monoclonal antibodies such asalemtuzumab, epratuzumab, gemtuzumab, ibritumomab tiuxetan, rituximab,tositumomab, and trastuzumab; protein kinase inhibitors such asbevacizumab, cetuximab, dasatinib, erlotinib, gefitinib, imatinib,lapatinib, mubritinib, nilotinib, panitumumab, pazopanib, sorafenib,sunitinib, and vandetanib; angiogeneisis inhibitors such as angiostatin,endostatin, bevacizumab, genistein, interferon alpha, interleukin-2,interleukin-12, pazopanib, pegaptanib, ranibizumab, rapamycin,thalidomide; and growth inhibitory polypeptides such as erythropoietin,interleukins (e.g., IL-1, IL-2, IL-3, IL-6), leukemia inhibitory factor,interferons, thrombopoietin, TNF-α, CD30 ligand, 4-1 BB ligand, andApo-1 ligand. Also included are pharmaceutically acceptable salts,acids, or derivatives of any of the above listed agents. The mode ofadministration of the chemotherapeutic agent can and will vary dependingupon the agent and the type of cancer. A skilled practitioner will beable to determine the appropriate dose of the chemotherapeutic agent.

The radiotherapeutic agent may include a radioisotope. Suitableradioisotopes include, without limit, Iodine-131, Iodine-125,Iodine-124, Lutecium-177, Phosphorous-132, Rhenium-186, Strontium-89,Yttrium-90, Iridium-192, and Samarium-153. Alternatively, theradiotherapeutic agent may include a high Z-element chosen from gold,silver, platinum, palladium, cobalt, iron, copper, tin, tantalum,vanadium, molybdenum, tungsten, osmium, iridium, rhenium, hafnium,thallium, lead, bismuth, gadolinium, dysprosium, holmium, and uranium.The appropriate dose of the radiotherapeutic agent may be determined bya skilled practitioner.

DEFINITIONS

When introducing elements of the embodiments described herein, thearticles “a”, “an”, “the” and “said” are intended to mean that there areone or more of the elements. The terms “comprising”, “including” and“having” are intended to be inclusive and mean that there may beadditional elements other than the listed elements.

The compounds described herein can exist in tautomeric, geometric orstereoisomeric forms. The present disclosure contemplates all suchcompounds, including cis- and trans-geometric isomers, E- andZ-geometric isomers, R- and S-enantiomers, diastereomers, d-isomers,l-isomers, the racemic mixtures thereof, and other mixtures thereof.Pharmaceutically acceptable salts of such tautomeric, geometric orstereoisomeric forms are also included within the invention. Compoundsof the present disclosure containing an asymmetrically substituted atommay be isolated in optically active or racemic form. All chiral,diastereomeric, racemic forms and all geometric isomeric forms of astructure are intended, unless the specific stereochemistry or isomericform is specifically indicated. The terms “cis” and “trans” (or “E” and“Z”), as used herein, denote a form of geometric isomerism in which twocarbon atoms connected by a double bond will each have a hydrogen atomon the same side of the double bond (“cis”) or on opposite sides of thedouble bond (“trans”).

The term “acyl,” as used herein alone or as part of another group,denotes the moiety formed by removal of the hydroxyl group from thegroup COOH of an organic carboxylic acid, e.g., RC(O)—, wherein R is R¹,R¹O—, R¹R²N—, or R¹S—, R¹ is hydrocarbyl, heterosubstituted hydrocarbyl,or heterocyclo, and R² is hydrogen, hydrocarbyl, or substitutedhydrocarbyl.

The term “acyloxy,” as used herein alone or as part of another group,denotes an acyl group as described above bonded through an oxygenlinkage (O), e.g., RC(O)O— wherein R is as defined in connection withthe term “acyl.”

The term “alkyl” as used herein describes groups which are preferablylower alkyl containing from one to eight carbon atoms in the principalchain and up to 20 carbon atoms. They may be straight or branched chainor cyclic and include methyl, ethyl, propyl, isopropyl, butyl, hexyl andthe like.

The term “alkenyl” as used herein describes groups which are preferablylower alkenyl containing from two to eight carbon atoms in the principalchain and up to 20 carbon atoms. They may be straight or branched chainor cyclic and include ethenyl, propenyl, isopropenyl, butenyl,isobutenyl, hexenyl, and the like.

The term “alkoxide” or “alkoxy” as used herein is the conjugate base ofan alcohol. The alcohol may be straight chain, branched, cyclic, andincludes aryloxy compounds.

The term “alkynyl” as used herein describes groups which are preferablylower alkynyl containing from two to eight carbon atoms in the principalchain and up to 20 carbon atoms. They may be straight or branched chainand include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and thelike.

The term “aromatic” as used herein alone or as part of another groupdenotes optionally substituted homo- or heterocyclic conjugated planarring or ring system comprising delocalized electrons. These aromaticgroups are preferably monocyclic (e.g., furan or benzene), bicyclic, ortricyclic groups containing from 5 to 14 atoms in the ring portion. Theterm “aromatic” encompasses “aryl” groups defined below.

The terms “aryl” or “Ar” as used herein alone or as part of anothergroup denote optionally substituted homocyclic aromatic groups,preferably monocyclic or bicyclic groups containing from 6 to 10 carbonsin the ring portion, such as phenyl, biphenyl, naphthyl, substitutedphenyl, substituted biphenyl, or substituted naphthyl.

The terms “carbocyclo” or “carbocyclic” as used herein alone or as partof another group denote optionally substituted, aromatic ornon-aromatic, homocyclic ring or ring system in which all of the atomsin the ring are carbon, with preferably 5 or 6 carbon atoms in eachring. Exemplary substituents include one or more of the followinggroups: hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl,acyloxy, alkenyl, alkenoxy, aryl, aryloxy, amino, amido, acetal,carbamyl, carbocyclo, cyano, ester, ether, halo, heterocyclo, hydroxyl,keto, ketal, phospho, nitro, and thio.

The terms “halogen” or “halo” as used herein alone or as part of anothergroup refer to chlorine, bromine, fluorine, and iodine.

The term “heteroatom” refers to atoms other than carbon and hydrogen.

The term “heteroaromatic” as used herein alone or as part of anothergroup denotes optionally substituted aromatic groups having at least oneheteroatom in at least one ring, and preferably 5 or 6 atoms in eachring. The heteroaromatic group preferably has 1 or 2 oxygen atoms and/or1 to 4 nitrogen atoms in the ring, and is bonded to the remainder of themolecule through a carbon. Exemplary groups include furyl, benzofuryl,oxazolyl, isoxazolyl, oxadiazolyl, benzoxazolyl, benzoxadiazolyl,pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl,pyrimidyl, pyrazinyl, pyridazinyl, indolyl, isoindolyl, indolizinyl,benzimidazolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl,carbazolyl, purinyl, quinolinyl, isoquinolinyl, imidazopyridyl, and thelike. Exemplary substituents include one or more of the followinggroups: hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl,acyloxy, alkenyl, alkenoxy, aryl, aryloxy, amino, amido, acetal,carbamyl, carbocyclo, cyano, ester, ether, halo, heterocyclo, hydroxyl,keto, ketal, phospho, nitro, and thio.

The terms “heterocyclo” or “heterocyclic” as used herein alone or aspart of another group denote optionally substituted, fully saturated orunsaturated, monocyclic or bicyclic, aromatic or non-aromatic groupshaving at least one heteroatom in at least one ring, and preferably 5 or6 atoms in each ring. The heterocyclo group preferably has 1 or 2 oxygenatoms and/or 1 to 4 nitrogen atoms in the ring, and is bonded to theremainder of the molecule through a carbon or heteroatom. Exemplaryheterocyclo groups include heteroaromatics as described above. Exemplarysubstituents include one or more of the following groups: hydrocarbyl,substituted hydrocarbyl, alkyl, alkoxy, acyl, acyloxy, alkenyl,alkenoxy, aryl, aryloxy, amino, amido, acetal, carbamyl, carbocyclo,cyano, ester, ether, halo, heterocyclo, hydroxyl, keto, ketal, phospho,nitro, and thio.

The terms “hydrocarbon” and “hydrocarbyl” as used herein describeorganic compounds or radicals consisting exclusively of the elementscarbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwiseindicated, these moieties preferably comprise 1 to 20 carbon atoms.

The “substituted hydrocarbyl” moieties described herein are hydrocarbylmoieties which are substituted with at least one atom other than carbon,including moieties in which a carbon chain atom is substituted (i.e.replaced) with a heteroatom such as nitrogen, oxygen, silicon,phosphorous, boron, or a halogen atom, and moieties in which the carbonchain comprises additional substituents. These moieties may includealkyl, alkoxy, acyl, acyloxy, alkenyl, alkenoxy, aryl, aryloxy, amino,amido, acetal, carbamyl, carbocyclo, cyano, ester, ether, halo,heterocyclo, hydroxyl, keto, ketal, phospho, nitro, and thio.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing from thescope of the invention defined in the appended claims.

EXAMPLES

The following examples illustrate various iterations of the invention.

Example 1 Synthesis of Dehydroleucodine Analogs

The sesquiterpene lactone dehydroleucodine (FIG. 2) was isolated fromGynoxys verrucosa Wedd by the following procedure: the air-dried partsof Gynoxys verrucosa (200 g) were extracted with ethyl acetate (EtOAc)(dynamic maceration for 5 h) at room temperature and concentrated underreduced pressure. The extract (14 g) was filtered through a reversephase C₁₈ column (LiChroprep Merck 25-40 μm) with a mixture MeOH/H₂O85:15, for the removal of chlorophylls. The filtrate (7 g) wasfractioned by column chromatography using a hexane-EtOAc gradient.Dehydroleucodine (DHL) was eluted in the hexane:EtOAc (85:15) fraction(0.7 g) and recrystallized from EtOAc as a white crystalline solid.

In an effort to develop more water-soluble compounds, dehydroleucodinederivatives were synthesized through a stereoselective conjugateaddition of a primary or secondary amine to the α-methylene-γ-lactonemoiety (FIG. 2). Thus, the exocyclic conjugated methylene in the lactonering of DHL was reacted with the appropriate primary or secondary aminein a one-step reaction. The general synthetic procedure was thefollowing: a solution of dehydroleucodine (0.22 mmol) in EtOH (6 mL) wastreated with various primary or secondary amines (4.4 mmol) in thepresence of triethylamine (Et₃N) and the mixture was stirred at roomtemperature overnight. The reaction mixture was evaporated under reducedpressure and the residue was further purified by silica gel flashchromatography to yield products 3-5. In all cases, a good yield(60-91%) of a single diastereomeric product was obtained. CompoundsDHL-proline (3), DHL-piperidine (4) and DHL-morpholine (5) were used toprovide a basis for SAR analysis.

DHL-proline (3) was obtained following the above general procedure inthe presence of L-proline, and then purified by flash chromatography(silica gel, 0-50% hexane/ethyl acetate) (Yield: 44.3 mg (60%)).DHL-proline was obtained as a white powder; it has the molecular formulaC₂₀H₂₅NO₅ based on the HRMS (m/z 360.1823 [M+H]⁺) and NMR data. The IR(ATR) spectrum showed absorption bands (v_(max)) at 3510 (COO-H st),1760 (C═O st γ lactone) and 1670 (α-β unsaturated C═O), 1645, 1633, 1613(C═C st) cm⁻¹. Complete ¹H and ¹³C NMR data is provided in Table 1. Theassignments of its ¹H and ¹³C NMR resonances have been done involvingtwo-dimensional experiments COSY, HMQC, ROESY and HMBC. The proton andcarbon resonances appeared with almost the same chemical shifts andsimilar multiplicities as in DHL, except for the signals for H-12 at δ2.25 and the signals corresponding to H-13 which appeared as a doubletat δ 1.27 in the ¹H NMR spectrum, suggesting the presence of a secondarymethyl group on the lactone ring. This was consistent with analogouschanges in the ¹³C NMR spectrum. Thus, a large (anti) H-8/H-12 ³Jcoupling, as well as a ROESY peak between H-12 and H-9, was observed,indicating that these hydrogens are in the same molecular face. Inaddition, a ROESY peak between H-12 and H-8 indicated that thesehydrogens also lie on the same face. Finally, the structure of compound3 was confirmed by X-ray crystallographic analysis, and a perspectiveORTEP plot is shown in FIG. 3. The crystal structure of DHL-prolineshows that the H atom has migrated to the N atom of this molecule. Thedifference electron density map clearly shows this H atom, and showsnothing at the COO group. Also, the H atoms of the included watermolecule were found by difference map.

TABLE 1 NMR Spectroscopy Data (500 MHz) of DHL-Proline Position δ_(H)δ_(C) HMBC COSY 1 170.3 2 6.17 135.8 196.4, 170.3, 3.58, 2.28 131.8,52.4, 19.9 3 196.4 4 131.7 5 153.0 6 2.46 37.4 153.0, 131.7, 2.33 2.3352.9, 25.6, 21.7 7 2.08 25.6 153.0, 84.6, 52.4, 37.4 2.46, 2.33, 1.44,1.44 153.0, 84.6, 52.4, 37.4 24.6, 2.33, 2.08 8 2.34 52.4 153.0, 131.7,52.9, 25.6, 21.7 9 3.75 84.6 173.0, 131.7, 52.4, 43.4, 29.2, 25.6, 24.23.50, 2.34 10 3.50 52.4 170.3, 131.7, 135.8, 84.6, 52.4 6.17, 3.75,[2.42]^(a), [2.25] 11 176.0 12 2.76 43.4 176.0 3.41, 3.21, 2.34 13 3.4152.8 176.0, 69.2, 54.7, 43.4 3.21, 2.76 3.21 176.0, 54.7, 52.4, 43.43.41, 2.76 14 2.28 19.9 170.3, 135.8, 52.4 6.17, 3.50 15 2.42 21.7153.0, 131.7, 37.4 3.50 16 3.66 54.7 69.2, 52.8, 29.4, 24.2 2.80, 2.02.80 52.8, 29.4, 24.2 3.66, 2.0 17 2.00 (2H) 24.2 69.2, 54.7, 29.4 3.66,2.8 18 2.32 24.4 153.0, 131.7, 52.9, 25.6, 21.7 2.23 19 3.73 69.2 2.32,2.23 20 173.0 ^(a)Numbers in square brackets denote weak long rangecorrelations

DHL-piperidine (4) was obtained following the above general procedure inthe presence of piperidine, and then purified by flash chromatography(silica gel, 0-80% hexane/ethyl acetate) (Yield: 29.40 mg (66%)).DHL-piperidine was isolated as a pale yellow solid with the molecularformula C₂₀H₂₇NO₃ based on the HSMR (m/z 330.2075 [M+H]⁺). Its UVspectrum in (MeOH) shows two peaks at λ (log ∈) 204 (3.66) nm and 256(4.14) nm. The IR (ATR) spectrum shows bands at 2936 (C—H st), 1773 (C═Ost γ lactone), 1683 (α-β unsaturated C═O), 1617, 1637 (C═C st) cm⁻¹. TheEI spectrum shows one main fragment at m/z 244 (100) [dehydroleucodine].The ¹H and ¹³C NMR chemical shifts are shown in Table 2. Assignments ofall proton and carbon resonances were achieved involving the 2D NMRtechniques COSY, ROESY and HMBC.

TABLE 2 NMR Spectroscopy Data (500 MHz) of DHL-piperidine Position δ_(H)δ_(C) HMBC COSY 1 170.1 2 6.16 135.6 196.1, 170.1, 132.0, 52.8, 20.03.43, 2.29, 6.16 3 196.1 4 132.0 5 152.8 6 2.43 37.9 152.8, 132, 26.5,54.8, 37.9 2.20, 2.40, 2.43 2.30 7 2.40 26.5 2.60 1.34 8 2.20 54.8 84.3,52.8 2.40, 2.41, 3.60, 3.43, 2.79 9 3.60 84.3 170.2, 132.0, 52.8, 20.06.16, 3.60, 3.43, 2.20, 2.29 10 3.42 52.8 196.1, 170.1, 152.8, 135.7,132.0, 3.60, 6.16, 3.43, 2.29, 2.41 84.3, 54.8 11 176.9 12 2.41 44.02.20, 2.60, 2.79 13 2.79 58.2 176.9, 54.8, 44.0, 176.9, 54.8, 44.0 2.41,2.20, 2.79, 2.40, 2.20 2.60 14 2.29 20.0 170.2, 152.6, 135.6, 131.9,52.8, 6.16, 3.43 26.2 15 2.43 21.7 3.4 16 2.32, 2.60, 2.79, 2.42, 2.60,2.79 17 1.54 26.2 55.0, 26.2, 24.3 2.42, 1.43 18 1.43 24.3 55.0, 26.21.54, 2.42 19 55.0, 26.2, 24.3 20 2.42 55.0 2.32, 2.60, 2.79, 2.42, 2.322.60, 2.79

DHL-morpholine (5) was obtained following the above general procedure inthe presence of morpholine, and then purified by flash chromatography(silica gel, 0-80% hexane/ethyl acetate) (Yield: 61.80 mg (91%)).DHL-morpholine was obtained as colorless crystals with mp 158-159° C.with the molecular formula assigned as C₁₉H₂₅NO₄ on the basis of itsHRMS data (m/z 332.1848 [M+H]⁺). Its UV spectrum (MeOH) shows two peaksat λ (log ∈) 204 (3.99) nm and 256 (4.28) nm. The IR (ATR) spectrumshows bands at 2930 (C—H st), 1770 (C═O st γ lactone), 1680 (α-βunsaturated C═O), 1640, 1610 (C═C st) and 1110 (C—O—C) cm⁻¹. The EIspectrum showed the molecular ion at (m/z 331.10) with low intensity(2), with other fragments at m/z (rel. int.) 300 (6) [M⁺-CH₃O], 288 (3)[M⁺-C], 244 (0.5) [C₁₅O₃H₁₆ ⁺], 100.1 (100) [C₄O₂H₅N⁺], 86 (9)[C₃O₂H₃N⁺], 71.1 (1) [C₄NH₈+] and 57.1[CNH₄ ⁺] (2). The ¹H and ¹³C NMRassignments are given in Table 3. For all protons and carbon resonanceswere achieved by COSY, ROESY, HSQC and HMBC experiments. The relativeconfiguration of compound 5 was determined by analysis of its NOESYdata. An NOE correlation was observed between H-8 and H-13 methyleneprotons but not H-12. H-8 shows three large (10-12 Hz) vicinal couplingsto H-9, H-12 and one of the H-7 (at delta 1.36) protons, indicating thatit is anti to H-9, H-12 and delta 1.36 proton. H-9 shows strong NOEpeaks to both H-12 and the delta 1.36 proton. Thus H-8 and H-12 are onopposite faces of the molecule. On the basis of this evidence, theabsolute stereochemistry of compound 5 was determined. Ultimately, thestructure of 5 was confirmed by X-ray crystallographic analysis, and aperspective ORTEP plot is shown in FIG. 4. The crystal unit cell iscomposed for two distinct molecular structures of DHL-morpholine, asshown in FIG. 5; one conformation is trans for C-13-N and C—H-12 bondswhile the other is gauche cis. These conformations are represented inFIG. 5.

TABLE 3 NMR Spectroscopic Data (500 MHz) of DHL-morpholine Positionδ_(H) δ_(C) HMBC COSY 1 169.9 2 6.17 (q, 1.3) 135.5 196.0, 169.9, 131.9,52.4, 19.7 3.43[2.29]^(a) 3 196.0 4 131.9 5 152.4 6 2.43 (m) 37.4 2.30(m) 7 2.34 (m) 26.1 152.4, 84.0, 54.1, 37.4 2.43, 2.34, 2.30, 1.36 (m)2.24 8 2.24 (m, −12, 54.1 3.62, 2.41, 2.34, 12, 10, 3) 1.36 9 3.62 (t,10.1) 84.0 131.9, 43.7, 26.1, 3.43, 2.24 10 3.43 (bd, 10) 52.4 196.0,169.9, 152.4, 135.5, 6.17, 3.62, [2.43]^(a), 131.7, 84.0, 54.1 [2.29] 11176.3 12 2.41 (m) 43.7 13 2.84 (dd, 13.2, 4.7) 57.2 176.3, 54.1, 43.7,176.3, 54.1, 2.63, 2.41, 2.84, 2.63 (dd, 13.2, 7.4) 43.7 2.41 14 2.29(3H, dd, 1.3, 0.9) 19.7 169.9, 135.5, 52.4 [6.17], [3.43] 15 2.43 (3H,bs) 21.3 152.4, 131.9, 37.4 [3.43] 16 2.51 (2H, m) 53.9 66.7, 53.9 2.432.43 (2H, m) 2.51 17 3.69 (4H, m) 66.7 66.7, 53.9 2.51, 2.43 18 3.69(4H, m) 66.7 66.7, 53.9 2.51, 2.43 19 2.51 (2H, m) 53.9 66.7, 53.9 2.432.43 (2H, m) 2.51 20 169.9 ^(a)Numbers in square brackets denotes weaklong range correlations.

DHL-tyramine: The compound was obtained by following the above generalprocedure in the presence of tyramine, and a mixture of EtOAc/Hexane 10%used as eluent in flash chromatography medium pressure.

Example 2 Conformations of DL-Morpholine

In order to determine which of the two morpholine conformations ispresent in solution, both conformers observed in the X-ray crystalstructure were analyzed. Thus, the vicinal hydrogens, one at C-12 andtwo at C-13 were considered for the measurements of dihedral angles. Thesingle crystal unit is composed of two molecules, each bearing theC-13/N bond of the morpholine moiety in opposite directions whilepreserving its chair conformation. The H-12/H-13 hydrogen-hydrogendihedral angles for each molecule that composes the crystal unit weremeasured using Mercury software, and these values were used to estimatethe coupling constants using a generalized Karplus curve of vicinalcouplings for protons; values are given in Table 4.

According to the couplings observed experimentally, the ²J coupling ofC-13 hydrogens is 13.2 Hz with further splittings of 7.4 and 4.7 Hz withH-12. This implies that the dominant average conformation is where H-12has one near anti plus a gauche relationship with the hydrogens at C-13,while the population having an almost equal gauche relationship of H-12to both C-13 hydrogens is negligible.

TABLE 4 Hydrogen-Hydrogen dihedral angles for Conformations 1 and 2Dihedral angle Angle (J)^(a) Fragment Conformation 1 Conformation 2H-12-C-12-C-13-H-13_(a) 162.53° (11 Hz) 61° (4 Hz) H-12-C-12-C-13-H-13_(b)   79.91° (2.5 Hz) −56° (4.5 Hz) ^(a)Estimatedusing Karplus curve

The atoms in a molecule can adopt many different positions(conformations) without undergoing a rearrangement of their chemicalbonds. Transformations from one configuration to another occur viarotation about a single bond with minor alterations of bond angle andlength. In order to find the lowest-energy conformations of a moleculethere are a number of methods available in SYBYL 8.1. Grid-Searchanalysis was used, which systematically searches a molecule withminimization of every conformation. The conformers were obtained byperforming systematic torsion angle changes around the C12 and C13single bond with 10-degree increments, using the Steepest Descendentmethod for the energy minimization with 1000 iterations and a gradientof 0.05. Starting geometries were optimized using MOPAC electrostaticcharges and TRIPOS force field, with dielectric function distance and8.0 as NB cutoff.

The results of the Grid-Search conformation analysis showed threeminimums and three maximum (FIG. 6). These minimums correspond with thestaggered conformations. Two of these three minimums, conformations Band F, with energies of 14.60 and 14.61 respectively, correspond withthe conformations displayed in the crystal structure unit cell. Thedifference in energy between these conformations is very small: about0.01 kcal/mol. The three maximums, conformations A, C and E withenergies of 21.00, 21.50 and 25.40 kcal/mol respectively, correspond tothe eclipsed conformations.

Example 3 Cytotoxic Activity of DHL and its Derivatives on Cancer Cells

In order to assess the structure-activity relationship of DHL and itsderivatives, DHL and its derivatives were evaluated against eight humanleukemia cell lines. Cell lines were cultured in Iscove's ModifiedDulbecco's Medium (IMDM) supplemented with 10%-20% fetal bovine serum(FBS) according to culture conditions indicated by ATCC and 1%penicillin/streptomycin (Pen/Strep) at 37° C. and 5% CO₂. The cells wereseeded into 96-well plates and kept at a concentration of 0.5 millioncells per mL. The samples were treated with DHL, Leucodine,DHL-Morpholine, DHL-Proline, DHL-Piperidine, and PTL at varyingconcentrations in triplicates. Cell viability was determined afterincubating for 48 hours. The cells were stained with annexinV-fluorescein isothiocyanate or phycoeritrine (FITC/PE) and7-aminoactinomycin (7-AAD) to detect phosphatidylserine exposition andcell permeability, respectively. At least 50,000 events were recordedper condition on either an LSR-II or LSR-Fortessa flow cytometer (BDBiosciences). Data analysis was conducted using FlowJo 9.6 software forMac OS X (TreeStar). Cells that were negative for annexin V and 7-AADwere scored as viable.

The results are shown in Table 5, with the inclusion of the extract G.Verrucosa as a reference. This is one of the first studies where DHL,another plant-derived SL, has been examined in AML. As seen from Table5, DHL has potent activity against multiple AML leukemic cell linesafter 48 hours of treatment, with LD₅₀ values ranging from 5.02-18.95 μMin the various leukemic cell lines tested. Each cell line was alsotested with PTL at 10 μM for comparison, with an average viability of70.4% after 48 hours. Thus, DHL displayed more potent anti-leukemicactivity than PTL in most samples. Leucodine was not found to be active,suggesting that the exocyclic methylene in DHL is necessary for theobserved activity. The in vitro cytotoxicity of the amino derivatives ofDHL is maintained, but at a more moderate level, with DHL-Prolineserving as the most potent derivative.

TABLE 5 Cytotoxic effects of DHL, its derivatives, and G. Verrucosaextract, expressed as LD₅₀ values (μM for DHL and derivatives of DHL,μg/mL for extract) DHL- DHL- DHL- G. Verrucosa DHL Leucodine ProlinePiperidine Morpholine Extract (μM) (μM) (μM) (μM) (μM) (μg/mL) HL-6014.13 >160 50.2 96.5 166 4.254 Kasumi-1 12.92 >160 28.1 >160 >160 3.546KG-1 18.66 >160 97.7 182 129 7.102 MOLM-13 12.59 >160 20.6 52.3 >1603.146 MV4-11 5.02 >160 23.3 66.9 21.1 2.98 THP-1 16.82 121 26.7 59.354.4 6.957 TUR 12.18 105 35.9 >160 >160 5.715 U-937 18.95 >160 33.0 >16074.7 6.012 Drug range tested: [1.25-160 μM]

Example 4 Cytotoxic Activity of DHL and its Derivatives on Normal Cells

When determining the potential of a new chemotherapeutic agent for usein patients, it is important that the drug effectively targets tumorcells without causing significant harm to noncancerous cells. In orderto better establish our understanding of DHL as an anticancer drug, wetested the activity of DHL and its derivatives on normal bone marrow(n=1) and peripheral blood (n=3) mononuclear cells. Bone marrow andperipheral blood samples were obtained from volunteer donors.Mononuclear cells were isolated from the samples using Ficoll-Plaquedensity gradient separation. Cells were cultured in serum-free medium(SFM) supplemented with cytokines (50 ng/ml rhFLT-3 ligand, 50 ng/mlrhSCF, 20 ng/ml rhIL-3, 20 ng/ml rhIL-6) for 1 h before the addition ofdrugs. The cells were seeded into 96-well plates and kept at aconcentration of 0.5 million cells per mL. The samples were treated withDHL, Leucodine, DHL-Morpholine, DHL-Proline, DHL-Piperidine, and PTL atvarying concentrations in triplicates. Cell viability was determinedafter incubating for 48 hours. The cells were stained with annexinV-fluorescein isothiocyanate or phycoeritrine (FITC/PE) and7-aminoactinomycin (7-AAD) to detect phosphatidylserine exposition andcell permeability, respectively. At least 50,000 events were recordedper condition on either an LSR-II or LSR-Fortessa flow cytometer (BDBiosciences). Data analysis was conducted using FlowJo 9.6 software forMac OS X (TreeStar). Cells that were negative for annexin V and 7-AADwere scored as viable.

FIG. 7A displays the viability of the normal samples after 48 hours oftreatment with either 20 μM DHL or 50 μM of the amino derivatives, withthe inclusion of PTL and G. verrucosa extract to serve as references.Since these SL's are derived from plants, we initially expected there tobe little toxicity. Leucodine and the amino derivatives display verylittle toxicity overall. DHL, however, was shown to have a similarcytotoxic effect as PTL. When compared to PTL, DHL was slightly lesstoxic to normal cells but more toxic in most of the AML cell lines.Importantly, FIG. 7B shows that DHL is significantly more potent to theAML cell lines tested (n=8) than to normal mononuclear cells (n=4,p=0.004). Thus, DHL has the cytotoxic profile to serve as a suitablechemotherapeutic agent.

Example 5 Mechanism of Action of DHL and its Derivatives

To better understand the mechanism by which DHL kills AML, theintracellular effects upon DHL treatment were evaluated. To comparethese intracellular effects of DHL and its derivatives, quantitative PCRwas performed (FIG. 8 and FIG. 9). MOLM-13 or MV-411 cells were seededat 0.5 million cells per milliliter. Cells were treated for 6 hours with20 μM of DHL, Leucodine, DHL-Morpholine, DHL-Proline, DHL-Piperidine orPTL, and then collected. Total RNA was extracted using the Qiashredderand Qiagen Rneasy Mini kits. The real-time PCR was performed using theTaqman RNA to CT 1-Step kit. The thermal cycling conditions were one RTstep (48° C., 15 minutes), Enzyme activation (95° C., 10 minutes) and 40cycles of Denature (95° C., 15 seconds) and Anneal/extend (60° C., 1minute). Quantitative PCR was performed using probes for HMOX-1(Hs01110251_m1), HSPA1A (Hs00359163_s1), and NFKB1 (Hs00765730_m1).GADPH (Hs02758991_g1) was used as housekeeping gene, an internal controlto normalize the variability in expression levels. Single-plex real-timePCR was performed in triplicates in a StepOne plus RealTime PCR systemand analyzed with the StepOne Software. Fold change was calculated usingthe 2-DDCT method described by Livak and Schmittgen.

HMOX1 (Heme oxygenase 1) was measured to compare the relative levels ofoxidative stress upon drug treatment (FIG. 8A and FIG. 9A). HSPA1A, themain stress-inducible isoform of HSP70, was also measured to compareoxidative stress (FIG. 8B and FIG. 9B). Although both DHL and PTLupregulated HMOX1 and HSPA1A, the higher fold changes by DHL treatmentsuggest that DHL may induce a greater amount of oxidative stress thanPTL. Leucodine and the other DHL amino derivatives upregulated HMOX1 andHSPA1A in both MOLM-13 cells and MV-411 cells, although to a lesserextent in MOLM-13 cells. Interestingly, DHL-proline, when compared tothe other DHL amino derivatives, upregulated HMOX1 and HSPA1A the mostand also displayed the greatest cytotoxic effect on the AML cell lines,as seen from Table 5.

PTL is a SL that has been shown to induce apoptosis on leukemic stem andprogenitor cells through inhibition of NF-κB.⁴ As a comparison, NF-κBtranscript levels were measured upon drug treatment in MOLM-13 cells(FIG. 8C) and MV-411 cells (FIG. 10). DHL, leucodine, and the DHL aminoderivatives slightly downregulated NF-κB, while PTL treatment displayedthe highest degree of downregulation.

In addition, the levels of phospho-p65 and total p65 by Western blotupon drug treatment of MOLM-13 cells was evaluated (FIG. 8D). Thetranscription factor p65 is involved in NF-κB heterodimer formation,with the phosphorylation of p65 being essential for NF-κB activation.MOLM-13 cells were seeded at 0.5 million cells per milliliter andsubject to either 10 or 20 μM of DHL or PTL treatment for 6 hours. Cellswere lysed and protein lysates were resolved on a 10% SDS-PAGE andtransferred to PVDF membrane. The membrane was incubated with primaryantibodies at 4° C. overnight, washed and incubated with IRDye 680 goatanti-rabbit or IRDye 800CW goat anti-mouse secondary antibodies at roomtemperature for 30 minutes. The membrane was then washed with phosphatebuffered saline with 0.1% Tween 20×. The membrane was scanned using theOdyssey infrared imaging system. Phospho-NF-κB p65 mouse primaryantibody and NF-κB p65 rabbit primary antibody were separately used tostain the membranes.

Strikingly, DHL and PTL both induced a significant decrease inphospho-p65. Total p65 is shown as a control. It is important to notethat a similar change in phospho-p65 with DHL treatment was observed inin MV4-11 cells (Data not shown).

Example 6 Effects of Dehydroleucodine and its Derivatives on GeneExpression

MOLM-13 and KG-1 cells were seeded at 0.5 million cells per ml. Cellswere treated for 6 hours with 20 μM of parthenolide (PTL),dehydroleucodine (DHL), leucodine (Leu), parthenolide-tyramine(PTL-Tyr), dehydroleucodine-tyramine (DHL-Tyr) anddehydroleucodine-Morpholine (DHL-Morp), and then collected. Total RNAwas extracted using the Qiashredder and Qiagen Rneasy mini kits(Quiagen) according to the manufacturer's instructions. Real-time PCRwas performed using the Taqman RNA to CT 1-Step kit (AppliedBiosystems). The thermal cycling conditions were one RT step (48° C., 15minutes), enzyme activation (95° C., 10 minutes) and 40 cycles ofdenature (95° C., 15 seconds) and anneal/extend (60° C., 1 minute).Real-time PCR was performed using probes for HMOX-1 (Hs01110251_m1),HSPA1A (Hs00359163_s1), or HSPH1 (Hs00971475_m1). GADPH (Hs02758991_g1)was used as an internal control to normalize the variability inexpression levels. All probes were provided by Applied Biosystems.Single-plex real-time PCR was performed in triplicates in a StepOne plusRealTime PCR system (Applied Biosystems) and analyzed with the StepOneSoftware. Fold change was calculated using the 2-DDCT method.

The changes in expression of HMOX-1, HSPA1A, and HSP1 in KG-1 cells areshown in FIGS. 1A, 1B, and 1C, respectively, and the changes inexpression of HMOX-1 and HSP1 in MOLM-13 cells are presented in FIGS. 1Dand 1E, respectively. DHL activated the expression of all three targetgenes in both cell lines, and DHL was more potent than PTL. Some of theDHL derivatives upregulated HSPH1 expression.

Example 7 Effects of Dehydroleucodine on Growth of Tumor Cells

As shown in Table 6, DHL inhibits tumor cells to varying degrees. DHL iscytotoxic to various leukemia, colon cancer, CNS cancer and prostatecancer cell lines. Surprisingly, DHL is not cytotoxic to variousnon-small cell lung cancer and ovarian cancer cell lines.

TABLE 6 Single dose results of dehydroleucodine tested at 10 μMPanel/Cell line Percent inhibition Panel cell lines for which DHL iscytotoxic Leukemia CCRF-CEM 44.9 K-562 42.0 RPMI-8226 31.7 Colon CancerHCT-116 41.3 SW-620 30.0 CNS Cancer SNB-75 35.5 U251 30.0 ProstateCancer PC-3 38.6 DU-145 44.0 Panel cell lines for which DHL is notcytotoxic Non-Small Cell Lung Cancer HOP-62 0.0 NCI-H322M 0.0 NCI-H4600.0 Ovarian Cancer NCI/ADR-RES 0.0 SK-OV-3 0.0

1. A compound comprising Formula (I) or a pharmaceutically acceptablesalt thereof:

wherein: R¹ and R² are independently hydrogen, hydrocarbyl, orsubstituted hydrocarbyl, or R¹ and R² together form an optionallysubstituted, saturated or unsaturated, carbocyclic or heterocyclic ringor ring system; R³ is hydrogen, hydroxy, amine, cyano, halo, nitro,phospho, sulfo, thiol, hydrocarbyl, or substituted hydrocarbyl; R⁴, R⁵,R⁶, R⁷, R⁸, and R⁹ are independently hydrogen, hydroxy, amine, cyano,halo, nitro, phospho, sulfo, thiol, hydrocarbyl, or substitutedhydrocarbyl; or any pair of R⁴ and R⁵, R⁶ and R⁷, or R⁸ and R⁹ togetherform ═O, ═S, ═CH₂, or ═NR^(a), wherein R^(a) is hydrogen, hydrocarbyl,or substituted hydrocarbyl; Z¹ and Z² are independently oxygen, sulfur,nitrogen, or CH₂; and

is a single or double bond.
 2. The compound of claim 1, wherein R¹ andR² are independently hydrogen, alkyl, substituted alkyl, heterocyclic,substituted heterocyclic, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, or R¹ and R² together form a heterocylic, substitutedheterocyclic, heteroaryl, or substituted heteroaryl ring or ring system.3. The compound of claim 1, wherein the compound comprising Formula (I)comprises Formula (Ia):

wherein: R¹ and R² are independently hydrogen, alkyl, substituted alkyl,cycloalkyl, haloalkyl, alkoxy, hydroxyalkyl, heterocyclic, substitutedheterocyclic, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, arylalkyl, heteroaryl, substitutedheteroaryl, carbonylalkyl, carbonyl substituted alkyl, carbonylalkoxy,carbonylaminoalkyl, carbonylaryl, carbonyl substituted aryl,carbonylaryloxy, or carbonylaminoaryl, or R¹ and R² together form aheterocylic, substituted heterocyclic, heteroaryl, or substitutedheteroaryl ring or ring system; R³, R⁶, and R⁸ are independentlyhydrogen, hydroxy, amine, cyano, halo, nitro, phospho, sulfo, thiol,hydrocarbyl, or substituted hydrocarbyl; and R⁴ and R⁵ are independentlyhydrogen, hydroxy, amine, cyano, halo, nitro, phospho, sulfo, thiol,hydrocarbyl, or substituted hydrocarbyl, or R⁴ and R⁵ together form ═O,═S, ═CH₂, or ═NR^(a), wherein R^(a) is hydrogen, hydrocarbyl, orsubstituted hydrocarbyl.
 4. The compound of claim 3, wherein R³ is C₁-C₆alkyl, R⁵ and R⁶ are hydrogen, and R⁸ is C₁-C₆ alkyl.
 5. The compound ofclaim 4, wherein R⁴ is —XR^(b), wherein X is —O—, —NH—, —S—, —SO—,—SO₂—, —CO—, —CO₂— and R^(b) is hydrogen, alkyl, substituted alkyl,haloalkyl, alkoxy, hydroxyalkyl, cycloalkyl, substituted cycloalkyl,heterocycloalkyl, substituted heterocycloalkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,arylalkyl, heteroaryl, substituted heteroaryl, carbonylaminoalkyl,carbonylamino substituted alkyl, carbonylaminoaryl, or carbonylaminosubstituted aryl.
 6. The compound of claim 4, wherein R¹ and R² togetherform an optionally substituted ring chosen from aziridinyl, azetidinyl,pyrrolidinyl, piperidynyl, or heptamethyleneiminyl.
 7. The compound ofclaim 1, wherein the compound comprising Formula (I) comprises Formula(Ib):

wherein: R¹ and R² are independently hydrogen, alkyl, substituted alkyl,cycloalkyl, haloalkyl, alkoxy, hydroxyalkyl, heterocyclic, substitutedheterocyclic, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, arylalkyl, heteroaryl, substitutedheteroaryl, carbonylalkyl, carbonyl substituted alkyl, carbonylalkoxy,carbonylaminoalkyl, carbonylaryl, carbonyl substituted aryl,carbonylaryloxy, or carbonylaminoaryl, or R¹ and R² together form aheterocylic, substituted heterocyclic, heteroaryl, or substitutedheteroaryl ring or ring system; R³, R⁴, and R⁶ are independentlyhydrogen, hydroxy, amine, cyano, halo, nitro, phospho, sulfo, thiol,hydrocarbyl, or substituted hydrocarbyl; and R⁸ and R⁹ are independentlyare independently hydrogen, hydroxy, amine, cyano, halo, nitro, phospho,sulfo, thiol, hydrocarbyl, or substituted hydrocarbyl, or R⁸ and R⁹together form ═O, ═S, ═CH₂, or ═NR^(a), wherein R^(a) is hydrogen,hydrocarbyl, or substituted hydrocarbyl.
 8. The compound of claim 7,wherein R³ is C₁-C₆ alkyl, R⁴ and R⁶ are hydrogen, and R⁶ is C₁-C₆alkyl.
 9. The compound of claim 8, wherein R⁹ is —XR^(c), wherein X is—O—, —NH—, —S—, —SO—, —SO₂—, —CO—, —CO₂— and R^(c) is hydrogen, alkyl,substituted alkyl, hydroxyalkyl, alkenyl, substituted alkenyl, alkynyl,or substituted alkynyl.
 10. The compound of claim 1, wherein thecompound comprising Formula (I) comprises Formula (Ic):

wherein: R¹ and R² are independently hydrogen, alkyl, substituted alkyl,cycloalkyl, haloalkyl, alkoxy, hydroxyalkyl, heterocyclic, substitutedheterocyclic, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, arylalkyl, heteroaryl, substitutedheteroaryl, carbonylalkyl, carbonyl substituted alkyl, carbonylalkoxy,carbonylaminoalkyl, carbonylaryl, carbonyl substituted aryl,carbonylaryloxy, or carbonylaminoaryl, or R¹ and R² together form aheterocylic, substituted heterocyclic, heteroaryl, or substitutedheteroaryl ring or ring system; R³ and R⁶ are independently hydrogen,hydroxy, amine, cyano, halo, nitro, phospho, sulfo, thiol, hydrocarbyl,or substituted hydrocarbyl; and R⁸ and R⁹ are independently areindependently hydrogen, hydroxy, amine, cyano, halo, nitro, phospho,sulfo, thiol, hydrocarbyl, or substituted hydrocarbyl, or R⁸ and R⁹together form ═O, ═S, ═CH₂, or ═NR^(a), wherein R^(a) is hydrogen,hydrocarbyl, or substituted hydrocarbyl.
 11. The compound of claim 10,wherein R³ and R⁸ are C₁-C₆ alkyl.
 12. The compound of claim 11, whereinR⁶ is hydrogen or hydroxy.
 13. The compound of claim 12, wherein R⁹ ishydrogen, alkyl, substituted alkyl, cycloalkyl, haloalkyl, alkoxy,aminoalkyl, hydroxyalkyl, sulfoxyalkyl, sulfonylalkyl, thioalkyl,heterocyclic, substituted heterocyclic, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, arylalkyl,aryloxy, aminoaryl, thioaryl, sulfoxyaryl, sulfonylaryl, heteroaryl,substituted heteroaryl, cyano, carboxy, alkylcarboxylate, arylcarboxylate, carboxamide, N-alkyl carboxamide, N-aryl carboxamide,N,N-dialkyl carboxamide, N,N-diaryl carboxamide, N-alky N-arylcarboxamide, carbonyl aminoalkyl, carbonylamino substituted alkyl,carbonylaminoaryl, carbonylamino substituted aryl, carbonylalkoxy, orcarbonylaryloxy.
 14. The compound of claim 1, wherein the compoundcomprising Formula (I) comprises Formula (Id):

wherein: R is N-hydrocarbyl or N-substituted hydrocarbyl; and R³, R⁶,and R⁸ are independently hydrogen, hydroxy, amine, cyano, halo, nitro,phospho, sulfo, thiol, hydrocarbyl, or substituted hydrocarbyl.
 15. Thecompound of claim 14, wherein R is N-alkyl, N-cycloalkyl, N-alkenyl,N-alkynyl, N-aryl, N-substituted alkyl, N-substituted cycloalkyl,N-substituted alkenyl, N-substituted alkynyl, or N-substituted aryl. 16.The compound of claim 15, wherein R⁶ is hydrogen, and R³ and R⁸ areC₁-C₆ alkyl.
 17. (canceled)
 18. A method for inhibiting growth of acancer cell, the method comprising contacting the cancer cell with anamount of a compound comprising Formula (I), or a pharmaceuticallyacceptable salt thereof, effective to inhibit growth of the cancer cell,the compound comprising Formula (I):

wherein: R¹ and R² are independently hydrogen, hydrocarbyl, orsubstituted hydrocarbyl, or R¹ and R² together form an optionallysubstituted, saturated or unsaturated, carbocyclic or heterocyclic ringor ring system; R³ is hydrogen, hydroxy, amine, cyano, halo, nitro,phospho, sulfo, thiol, hydrocarbyl, or substituted hydrocarbyl; R⁴, R⁵,R⁶, R⁷, R⁸, and R⁹ are independently hydrogen, hydroxy, amine, cyano,halo, nitro, phospho, sulfo, thiol, hydrocarbyl, or substitutedhydrocarbyl; or any pair of R⁴ and R⁵, R⁶ and R⁷, or R⁸ and R⁹ togetherform ═O, ═S, ═CH₂, or ═NR^(a), wherein R^(a) is hydrogen, hydrocarbyl,or substituted hydrocarbyl; Z¹ and Z² are independently oxygen, sulfur,nitrogen, or CH₂; and

is a single or double bond. 19.-31. (canceled)
 32. The method of claim18, wherein the cancer cell is a leukemic cell. 33.-36. (canceled) 37.The method of claim 18, wherein the cancer cell is a solid tumor cell.38. The method of claim 37, wherein the solid tumor cell is selectedfrom the group consisting of a colon cancer cell, a prostate cancer celland a central nervous system cancer cell. 39.-48. (canceled)